Coli Surrogates Research Articles

A novel bicyclic 2,4-diaminopyrimidine inhibitor of Streptococcus suis dihydrofolate reductase.

Streptococcus suis is a Gram-positive bacterial pathogen of pigs and an emerging zoonotic pathogen. It has become increasingly resistant to multiple classes of antibiotics. New drug candidates and knowledge of their targets are needed to combat antibiotic-resistant S. suis. In this study, the open-source Pathogen Box compound library was screened. Thirty hits that effectively inhibited S. suis growth at 10 µM were identified. Among the most potent hits, MMV675968 (a diaminoquinazoline analog) was shown to target S. suis dihydrofolate reductase (SsDHFR) via (1) growth inhibition of an E. coli surrogate whose growth is dependent on exogenously expressed SsDHFR and (2) inhibition of in vitro SsDHFR activity. Thymidine supplement is able to reverse growth inhibition by MMV675968 in both E. coli surrogate and S. suis, indicating that a thymidine-related pathway is a major target of MMV675968. Comparison of MMV675968 with seven DHFR inhibitors representing different core structures revealed that bicyclic 2,4-diaminopyrimidines with long and flexible side chains are highly effective in inhibiting SsDHFR and S. suis growth. MMV675968 and related compounds thus may serve as starting points for developing antibiotics against drug resistant S. suis.

Persistence of Escherichia coli O157:H12 and Escherichia coli K12 as Non-pathogenic Surrogates for O157:H7 on Lettuce Cultivars Irrigated With Secondary-Treated Wastewater and Roof-Collected Rain Water in the Field

Treated wastewater (TW) and roof-collected rain water (RW) that meet the required microbial quality as per Food Safety Modernization Act (FSMA) regulation may serve as alternative irrigation water sources to decrease the pressure on the current water scarcity. Alternative water sources may have different water characteristics that influence the survival and transfer of microorganisms to the irrigated produce. Further, these water sources may contain pathogenic bacteria such as Shiga-toxigenicEscherichia coli. To evaluate the risk associated with TW and RW irrigation on the fresh produce safety, the effect of TW and RW irrigation on the transfer of two non-pathogenicE. colistrains as surrogates forE. coliO157:H7 to different lettuce cultivars grown in the field was investigated. Lettuce cultivars “Annapolis,” “Celinet,” and “Coastline” were grown in the field at the Fulton farm (Chambersburg, PA). Approximately 10 days before harvest, lettuce plants were spray-irrigated with groundwater (GW), TW, or RW containing 6 log CFU ml−1of a mixture of nalidixic acid-resistantE. coliO157:H12 and chloramphenicol-resistantE. coliK12 in fecal slurry as non-pathogenic surrogates forE. coliO157:H7. On 0, 1, 3, 7, and 10 days post-irrigation, four replicate lettuce leaf samples (30 g per sample) from each group were collected and pummeled in 120 ml of buffered peptone water for 2 min, followed by spiral plating on MacConkey agars with antibiotics. Results showed that the recovery ofE. coliO157:H12 was significantly greater than the populations ofE. coliK12 recovered from the irrigated lettuce regardless of the water sources and the lettuce cultivars. The TW irrigation resulted in the lowest recovery of theE. colisurrogates on the lettuce compared to the populations of these bacteria recovered from the lettuce with RW and GW irrigation on day 0. The difference in leaf characteristics of lettuce cultivars significantly influenced the recovery of these surrogates on lettuce leaves. Populations ofE. coliO157:H12 recovered from the RW-irrigated “Annapolis” lettuce were significantly lower than the recovery of this bacterium from the “Celinet” and “Coastline” lettuce (P< 0.05). Overall, the recovery of specificE. colisurrogates from the RW and TW irrigated lettuce was comparable to the lettuce with the GW irrigation, where GW served as a baseline water source.E. coliO157:H12 could be a more suitable surrogate compared toE. coliK12 because it is an environmental watershed isolate. The findings of this study provide critical information in risk assessment evaluation of RW and TW irrigation on lettuce in Mid-Atlantic area.

Diaminoquinazoline MMV675968 from Pathogen Box inhibits Acinetobacter baumannii growth through targeting of dihydrofolate reductase

Antibiotic resistance in Acinetobacter baumannii is a major global health threat. New drugs with novel chemical structures are needed to overcome a myriad of resistance mechanisms in A. baumannii. In this study, we screened an open-source Pathogen Box library for anti-A. baumannii compounds. Compound MMV675968 (a diaminoquinazoline analog) was the only non-reference compound found to inhibit the growth of all four A. baumannii test strains with IC50 of 0.6–2.7 μM, IC90 of 0.7–3.9 μM, and MIC of 1.6–10 μM. We showed that MMV675968 targeted A. baumannii dihydrofolate reductase (AbDHFR) as determined by an E. coli surrogate whose growth was dependent on AbDHFR function and by an in vitro DHFR activity assay. Additionally, chemical scaffolds of DHFR inhibitors that are effective as antibiotics against A. baumannii were identified using an in vitro DHFR activity assay and A. baumannii growth inhibition. MMV675968 was the most potent among DHFR inhibitors tested in inhibiting A. baumannii growth. This study shows for the first time that MMV675968 inhibits A. baumannii growth via selective inhibition of AbDHFR and is therefore a promising scaffold for further antibiotic development against A. baumannii.

Evaluation of thermal process lethality for non-pathogenic Escherichia coli as a surrogate for Salmonella in ground beef

The United States Department of Agricultures’ Food Safety and Inspection Service (USDA-FSIS) has developed thermal lethality guidelines for non-typhoidal Salmonella inactivation in ready-to-eat (RTE) beef and poultry, but additional means of thermal processing validation are limited. Thus, the objective of this study was to determine if non-pathogenic Escherichia coli could be used as a surrogate for Salmonella as means to validate thermal processing parameters per Appendix A. To develop thermal death time curves, ground beef at varying fat contents (5, 10, 20, 25, and 30%) was inoculated with either Salmonella or E. coli and heat treated. At 54, 57, 60, and 63 °C across all fat levels, the E. coli surrogates had significantly greater (P < .05) decimal-reduction values (D-values) than Salmonella. Beyond temperature 63 °C, regardless of fat, E. coli surrogates and Salmonella were inactivated at similar rates (P > .05). Greater reduction of E. coli surrogates in the ground beef post-lethality treatment suggest Salmonella inactivation at higher temperatures. The most appropriate use of the E. coli surrogates would be for predicting, ensuring, and validating thermal processing for Salmonella inactivation at lower temperatures. However, effects of meat product composition and processing facility variables need to be further assessed.

Characterization of the Serralysin-Like Gene of 'Candidatus Liberibacter solanacearum' Associated with Potato Zebra Chip Disease.

The nonculturable bacterium 'Candidatus Liberibacter solanacearum' is the causative agent of zebra chip disease in potato. Computational analysis of the 'Ca. L. solanacearum' genome revealed a serralysin-like gene based on conserved domains characteristic of genes encoding metalloprotease enzymes similar to serralysin. Serralysin and other serralysin family metalloprotease are typically characterized as virulence factors and are secreted by the type I secretion system (T1SS). The 'Ca. L. solanacearum' serralysin-like gene is located next to and divergently transcribed from genes encoding a T1SS. Based on its relationship to the T1SS and the role of other serralysin family proteases in circumventing host antimicrobial defenses, it was speculated that a functional 'Ca. L. solanacearum' serralysin-like protease could be a potent virulence factor. Gene expression analysis showed that, from weeks 2 to 6, the expression of the 'Ca. L. solanacearum' serralysin-like gene was at least twofold higher than week 1, indicating that gene expression stays high as the disease progresses. A previously constructed serralysin-deficient mutant of Serratia liquefaciens FK01, an endophyte associated with insects, as well as an Escherichia coli lacking serralysin production were used as surrogates for expression analysis of the 'Ca. L. solanacearum' serralysin-like gene. The LsoA and LsoB proteins were expressed as both intact proteins and chimeric S. liquefaciens-'Ca. L. solanacearum' serralysin-like proteins to facilitate secretion in the S. liquefaciens surrogate and as intact proteins or as a truncated LsoB protein containing just the putative catalytic domains in the E. coli surrogate. None of the 'Ca. L. solanacearum' protein constructs expressed in either surrogate demonstrated proteolytic activity in skim milk or zymogram assays, or in colorimetric assays using purified protein, suggesting that the 'Ca. L. solanacearum' serralysin-like gene does not encode a functional protease, or at least not in our surrogate systems.

Evaluating the Efficacy of Three U.S. Department of Agriculture–Approved Antimicrobial Sprays for Reducing Shiga Toxin–Producing Escherichia coli Surrogate Populations on Bob Veal Carcasses

Effective antimicrobial intervention strategies to reduce Shiga toxin-producing Escherichia coli (STEC) risks associated with veal are needed. This study evaluated the efficacy of lactic acid (4.5%, pH 2.0), Citrilow (pH 1.2), and Beefxide (2.25%, pH 2.3) for reducing STEC surrogates on prerigor and chilled bob veal carcasses and monitored the effects of these interventions on chilled carcass color. Dehided bob veal carcasses were inoculated with a five-strain cocktail of rifampin-resistant, surrogate E. coli bacteria.E. coli surrogates were enumerated after inoculation, after water wash, after prechill carcass antimicrobial spray application, after chilling for 24 h, and after postchill carcass antimicrobial spray application; carcass color was measured throughout the process. A standard carcass water wash (∼50°C) reduced the STEC surrogate population by 0.9 log CFU/cm(2) (P ≤ 0.05). All three antimicrobial sprays applied to prerigor carcasses delivered an additional ∼0.5-log reduction (P ≤ 0.05) of the surrogates. Chilling of carcasses for 24 h reduced (P ≤ 0.05) the surrogate population by an additional ∼0.4 log cycles. The postchill application of the antimicrobial sprays provided no further reductions. Carcass L*, a*, and b* color values were not different (P > 0.05) among carcass treatments. Generally, the types and concentrations of the antimicrobial sprays evaluated herein did not negatively impact visual or instrumental color of chilled veal carcasses. This study demonstrates that warm water washing, followed by a prechill spray treatment with a low-pH chemical intervention, can effectively reduce STEC risks associated with veal carcasses; this provides processors a validated control point in slaughter operations.

Antimicrobial Efficacy of a Lactic Acid and Citric Acid Blend against Shiga Toxin-Producing Escherichia coli, Salmonella, and Nonpathogenic Escherichia coli Biotype I on Inoculated Prerigor Beef Carcass Surface Tissue

Studies were conducted to (i) determine whether inoculants of nonpathogenic Escherichia coli biotype I effectively served as surrogates for E. coli O157:H7, non-O157 Shiga toxin-producing E. coli, and Salmonella when prerigor beef carcass tissue was treated with a commercially available blend of lactic acid and citric acid (LCA) at a range of industry conditions of concentration, temperature, and pressure; (ii) determine the antimicrobial efficacy of LCA; and (iii) investigate the use of surrogates to validate a hot water and LCA sequential treatment as a carcass spray intervention in a commercial beef harvest plant. In an initial laboratory study, beef brisket tissue samples were left uninoculated or were inoculated (∼6 log CFU/cm(2)) on the adipose side with E. coli O157:H7 (5-strain mixture), non-O157 Shiga toxin-producing E. coli (12-strain mixture), Salmonella (6-strain mixture), or nonpathogenic E. coli (5-strain mixture). Samples were left untreated (control) or were treated with LCA, in a spray cabinet, at one of eight combinations of solution concentration (1.9 and 2.5%), solution temperature (43 and 60°C), and application pressure (15 and 30 lb/in(2)). In a second study, the E. coli surrogates were inoculated (∼6 log CFU/cm(2)) on beef carcasses in a commercial facility to validate the use of a hot water treatment (92.2 to 92.8°C, 13 to 15 lb/in(2)) followed by an LCA treatment (1.9%, 50 to 51.7°C, 13 to 15 lb/in(2), 10 s). In the in vitro study, surrogate and pathogen bacteria did not differ in their response to the tested LCA treatments. Treatment with LCA reduced (P < 0.05) inoculated populations by 0.9 to 1.5 log CFU/cm(2), irrespective of inoculum type. The hot water and LCA sequential treatments evaluated in the commercial facility reduced (P < 0.05) the inoculated nonpathogenic E. coli surrogates on carcasses by 3.7 log CFU/cm(2). This study therefore provides the meat industry with data for this sequential multiple hurdle system for the operation parameters described.

Survival of Escherichia coli on strawberries grown under greenhouse conditions

Strawberries are soft fruit that are not recommended to have a post-harvest wash due to quality concerns. Escherichia coli O157:H7 has been linked to outbreaks with strawberries but little is known about the survival of E. coli during the growth cycle of strawberries. The survival of E. coli on strawberry plants during growing under greenhouses conditions was evaluated. Soil, leaves, and strawberries (if present) were artificially contaminated with an E. coli surrogate either at the time of planting, first runner removal (4 wk), second runner removal (8 wk), or one week prior to harvest. At harvest E. coli was recovered from the leaves, soil, and strawberries regardless of the contamination time. Time of contamination influenced (P < 0.05) numbers of viable E. coli on the plant. The highest survival of E. coli (P < 0.0001) was detected in soil that was contaminated at planting (4.27 log10 CFU g soil−1), whereas, the survival of E. coli was maximal at later contamination times (8 wk and 1 wk prior to harvest) for the leaves (4.40 and 4.68 log10 CFU g leaves−1) and strawberries (3.37 and 3.53 log10 CFU strawberry−1). Cross contamination from leaves to fruit was observed during this study, with the presence of E. coli on strawberries which had not been present at the time of contamination. These results indicate that good agricultural best practices to avoid contamination are necessary to minimize the risk of contamination of these popular fruit with enteric pathogens. Practices should include soil testing prior to harvest and avoiding contamination of the leaves.

Evaluation of Lactic Acid as an Initial and Secondary Subprimal Intervention for Escherichia coli O157:H7, Non-O157 Shiga Toxin–Producing E. coli, and a Nonpathogenic E. coli Surrogate for E. coli O157:H7

Lactic acid can reduce microbial contamination on beef carcass surfaces when used as a food safety intervention, but effectiveness when applied to the surface of chilled beef subprimal sections is not well documented. Studies characterizing bacterial reduction on subprimals after lactic acid treatment would be useful for validations of hazard analysis critical control point (HACCP) systems. The objective of this study was to validate initial use of lactic acid as a subprimal intervention during beef fabrication followed by a secondary application to vacuum-packaged product that was applied at industry operating parameters. Chilled beef subprimal sections (100 cm(2)) were either left uninoculated or were inoculated with 6 log CFU/cm(2) of a 5-strain mixture of Escherichia coli O157:H7, a 12-strain mixture of non-O157 Shiga toxin-producing E. coli (STEC), or a 5-strain mixture of nonpathogenic (biotype I) E. coli that are considered surrogates for E. coli O157:H7. Uninoculated and inoculated subprimal sections received only an initial or an initial and a second "rework" application of lactic acid in a custombuilt spray cabinet at 1 of 16 application parameters. After the initial spray, total inoculum counts were reduced from 6.0 log CFU/cm(2) to 3.6, 4.4, and 4.4 log CFU/cm(2) for the E. coli surrogates, E. coli O157:H7, and non-O157 STEC inoculation groups, respectively. After the second (rework) application, total inoculum counts were 2.6, 3.2, and 3.6 log CFU/cm(2) for the E. coli surrogates, E. coli O157:H7, and non-O157 STEC inoculation groups, respectively. Both the initial and secondary lactic acid treatments effectively reduced counts of pathogenic and nonpathogenic strains of E. coli and natural microflora on beef subprimals. These data will be useful to the meat industry as part of the HACCP validation process.