Two homologous Salmonella serogroup C1-specific genes are required for flagellar motility and cell invasion

Background Salmonella is a major bacterial pathogen associated with a large number of outbreaks of foodborne diseases. Many highly virulent serovars that cause human illness belong to Salmonella serogroup C1, and Salmonella ser. Choleraesuis is a prominent cause of invasive infections in Asia. Comparative genomic analysis in our previous study showed that two homologous genes, SC0368 and SC0595 in Salmonella ser. Choleraesuis were unique to serogroup C1. In this study, two single-deletion mutants (Δ0368 and Δ0595) and one double-deletion mutant (Δ0368Δ0595) were constructed based on the genome. All these mutants and the wild-type strain were subjected to RNA-Seq analysis to reveal functional relationships of the two serogroup C1-specific genes. Results Data from RNA-Seq indicated that deletion of SC0368 resulted in defects in motility through repression of σ28 in flagellar regulation Class 3. Consistent with RNA-Seq data, results from transmission electron microcopy (TEM) showed that flagella were not present in △0368 and △0368△0595 mutants resulting in both swimming and swarming defects. Interestingly, the growth rates of two non-motile mutants △0368 and △0368△0595 were significantly greater than the wild-type, which may be associated with up-regulation of genes encoding cytochromes, enhancing bacterial proliferation. Moreover, the △0595 mutant was significantly more invasive in Caco-2 cells as shown by bacterial enumeration assays, and the expression of lipopolysaccharide (LPS) core synthesis-related genes (rfaB, rfaI, rfaQ, rfaY, rfaK, rfaZ) was down-regulated only in the △0368△0595 mutant. In addition, this study also speculated that these two genes might be contributing to serotype conversion for Salmonella C1 serogroup based on their apparent roles in biosynthesis of LPS and the flagella. Conclusion A combination of biological and transcriptomic (RNA-Seq) analyses has shown that the SC0368 and SC0595 genes are involved in biosynthesis of flagella and complete LPS, as well as in bacterial growth and virulence. Such information will aid to revealing the role of these specific genes in bacterial physiology and evolution within the serogroup C1.

Medical Tape Contamination Study: Effect of Packaging on the Reduction of Cross Contamination

Background: Medical tape is used routinely for a variety of tasks across healthcare settings. The literature contains numerous publications in which common practices around medical tapes have been suspected to lead to infection transmission. Healthcare providers can turn to individually packaged single-patient-use medical tape rolls to help reduce cross-contamination risk by limiting exposure to environmental contaminants, minimizing contact with hospital surfaces and equipment, and minimizing exposure to healthcare workers’ hands and other patients. Methods: We evaluated the effect of individually packaged tape on cross contamination using a controlled laboratory assay. Ceramic tiles were inoculated with microorganisms evenly spread across the surface and allowed to air dry. Using gloves, packaged and unpackaged tapes were rolled over their entire outside circumference onto the contaminated tiles to simulate cross contamination. Using new gloves, the packaged tapes were then removed from their package with minimum contact. All cross-contaminated tape rolls were placed in phosphate-buffered water and mixed in a vortexer for bacterial recovery procedures. Serial dilutions were plated on appropriate media for bacterial enumeration. The average log10 colony-forming unit (CFU) recovery was measured for comparison. We used 4 types of tapes in this study (3M Micropore S Surgical Tape, 3M Medipore H Soft Cloth Surgical Tape, 3M Durapore Surgical Tape, and 3M Transpore White Surgical Tape). We used 4 different microorganisms as inoculates: Staphylococcus aureus (methicillin-resistant), Enterococcus faecium (vancomycin-resistant), Klebsiella pneumoniae (carbapenem-resistant), and Clostridium difficile (spore). Each test (tape and bacteria combination) was done in 3 or 6 replicates; each bacterial enumeration was the average of duplicate plates. The detection limit for this method is 8 CFU per sample, which is equivalent to 0.9 log10. Results: The results for all tapes tested showed a statistically significant lower mean log10 recovery of each of the microorganisms tested for packaged versus unpackaged tape (Figure 1). The mean differences of log10 recoveries from a packaged and unpackaged tape ranged from 2.51 log10 (for S. aureus on Micropore S) to 4.64 log10 (for K. pneumoniae on Medipore H). This is equivalent to 99%–99.99% cross-contamination protection from the 4 organisms tested. Conclusions: Individual packaging of medical tape rolls protects them from external contaminants. Even if the packaging becomes contaminated, the tape retrieved from the package will be significantly less contaminated than it would have been from exposure to the same contaminants without packaging.Funding: 3M CompanyDisclosures: None

Microbial Growth Study on Pork Loins as Influenced by the Application of Different Antimicrobials

The use of antimicrobials in the pork industry is critical in order to ensure food safety and, at the same time, extend shelf life. The objective of the study was to determine the impact of antimicrobials on indicator bacteria on pork loins under long, dark, refrigerated storage conditions. Fresh boneless pork loins (n = 36) were split in five sections and treated with antimicrobials: Water (WAT), Bovibrom 225 ppm (BB225), Bovibrom 500 ppm (BB500), Fit Fresh 3 ppm (FF3), or Washing Solution 750 ppm (WS750). Sections were stored for 1, 14, 28, and 42 days at 2–4 °C. Mesophilic and psychrotrophic aerobic bacteria (APC-M, APC-P), lactic acid bacteria (LAB-M), coliforms, and Escherichia coli were enumerated before intervention, after intervention, and at each storage time. All bacterial enumeration data were converted into log10 for statistical analysis, and the Kruskal–Wallis test was used to find statistical differences (p < 0.05). Initial counts did not differ between treatments, while, after treatment interventions, treatment WS750 did not effectively reduce counts for APC-M, APC-P, and coliforms (p < 0.01). BB500, FF3, and WS750 performed better at inhibiting the growth of indicator bacteria when compared with water until 14 days of dark storage.

Interpretation and Implications of Lognormal Linear Regression Used for Bacterial Enumeration

Background Bacterial enumeration data are typically log transformed to realize a more normal distribution and stabilize the variance. Unfortunately, statistical results from log transformed data are often misinterpreted as data within the arithmetic domain. Objective To explore the implication of slope and intercept from an unweighted linear regression and compare it to the results of the regression of log transformed data. Method Mathematical formulae inferencing explained using real dataset. Results For y=Ax+B+ε, where y is the recovery (CFU/g) and x is the target concentration (CFU/g) with error ε homogeneous across x. When B=0, slope A estimates percent recovery R. In the regression of log transformed data, logy=αlogx+β+εz (equivalent to equation y=Axα·ω), it is the intercept β=logyx=logA that estimates the percent recovery in logarithm when slope α=1, which means that R doesn’t vary over x. Error term ω is multiplicative to x, while εz or log(ω) is additive to log(x). Whether the data should be transformed or not is not a choice, but a decision based on the distribution of the data. Significant difference was not found between the five models (the linear regression of log transformed data, three generalized linear models and a nonlinear model) regarding their predicted percent recovery when applied to our data. An acceptable regression model should result in approximately the best normal distribution of residuals. Conclusions Statistical procedures making use of log transformed data should be studied separately and documented as such, not collectively reported and interpreted with results studied in arithmetic domain. Highlights The way to interpret statistical results developed from arithmetic domain does not apply to that of the log transformed data.

Effect of Organic Acid Applications on Organoleptic Properties of Ground Pork

ObjectivesThe objective of this study was to determine the impact of intervention treatments on the organoleptic properties of ground pork during shelf-life.Materials and MethodsPork trimmings were divided into 22 kg batches for each individual treatment (n = 4 batches). Treatments included control (no intervention), PAA+Titon [Sulfuric acid and sodium sulfate (pH 1.3) combined with peracetic acid (350 ppm)], PAA+Acetic [Peracetic acid (400 ppm) with 2% acetic acid], and LA [Lactic acid (3%)]. After application of each designated treatment, trimmings were ground (coarse and then fine ground) and packaged into 454-g vacuum packaged rollstock packaging. Each package was then stored in dark storage for 0, 7, 14, 21, and 28 d at 2–4°C (n = 8 packages per treatment and time combination). Once each package had reached their designated storage length, packages were removed from storage for sampling. Shelf-life measurements taken included TBARS, raw product odor acceptability, aerobic plate count and psychrotrophic plate count bacterial enumeration. All bacterial enumeration data were converted into log10 for statistical analysis, and the PROC MIXED procedure of SAS was used to determine differences between least squared means (SAS Inst. Inc., Version 9.4, Cary, NC). Odor acceptability was determined using a PROC FREQ.ResultsFor aerobic plate counts (APC), in the ground pork product, a treatment by day interaction occurred (P = 0.007). Psychrotrophic bacterial counts did not differ by treatment or sampling day (P > 0.05). PAA+Titon and LA had decreased lipid oxidation compared to PAA+Acetic and control pork samples over the 28 d of storage. Lipid oxidation didn’t change for all 4 treatments on Days 0, 14, 21, and 28; however, there was an unexplainable spike in lipid oxidation for samples on Day 7. Overall pork odor acceptability differed by storage length (P = 0.02), but not by treatment (P > 0.05). Overall Pork odor acceptability decreased as storage length increased. Acidic off-odor differed by storage length (P = 0.002), but not by treatment (P > 0.05). Acidic off-odor increased as storage length increased. Overall oxidation off-odor did not differ by treatment or aging (P > 0.05). Sweaty off-odor development differed by aging day (P = 0.01) but not by treatment (P > 0.05). Sweaty off-odor reached its highest point within Day 14 and 21 and then decreased. An increasing sour off-odor development differed by ground pork storage length (P = 0.001), but not by treatment (P > 0.05).ConclusionThere were no dramatic negative organoleptic changes to pork trim when treated with selected organic acid interventions prior to grinding, meanwhile there is organoleptic changes by storage length.

Effects of Bacteriophage, Ultraviolet Light, and Organic Acid Applications on STEC O157:H7 and the “Big Six” in Beef Packaged Under Vacuum and Aerobic Conditions

ObjectivesBeef primals produced during high event periods (HEP) can also be affected by STEC contamination requiring microbial assessment. Commonly, primals are retreated with antimicrobials after removal from vacuum bags, then repackaged and tested for STEC. In this study, we evaluated the efficiency of bacteriophage, ultraviolet light, and organic acids on contaminated beef kept under vacuum and aerobic conditions.Materials and MethodsThe effects of antimicrobial interventions Peroxyacetic acid (PAA, 400 PPM), Ultraviolet light (UV, 30 s at 2.5 ± 0.3 cm height), Acidified Sodium Chlorite (ASC, 1200 ppm), and bacteriophage (P, 7 MS phages at 108 PFU/ml) against STEC (O157:H7 and O145, O121, O111, O103, O45, O26) were evaluated on beef. Fresh m. cutaneous trunci was fabricated into 100 cm2 samples (n = 154), which were randomly assigned to 11 treatments including Control, P, UV, ASC, PAA, and combinations P+UV, P+PAA, P+ASC, UV+PAA, UV+ASC, PAA+ASC. Treatments were tested under vacuum and aerobic conditions. Samples were inoculated with a STEC cocktail comprised of 7 strains to yield 3 log CFU/cm2. Samples were vacuumed or overwrapped with oxygen permeable film. Samples were unpackaged and treated with buffered peptone water (BPW, Control) or individual or combined antimicrobial treatments prior to re-packaging. After 1 h at 7°C, samples were swabbed, homogenized in 1 mL of BPW, serially diluted and spread-plated for bacterial enumeration. Data was analyzed using SAS as a completely randomized design.ResultsOverall, treatments including MS phages significantly decreased STEC populations in beef under vacuum and aerobic conditions (P < 0.0001). Under vacuum, individual phage application, combinations between phage and UV, ASC, and PAA plus UV+ASC provided optimal STEC reduction on beef surface. Phage and PAA combination led to the lowest STEC load (1.49 log reduction). When analyzing contrasts, treatments with phage significantly decreased STEC loads when compared to other treatments (P < 0.0001) and control (P < 0.0001). STEC loads recovered from treatments without phage and control were statistically similar at P = 0.32. Under aerobic conditions, individual treatments UV and ASC and combinations including UV+PAA, and PAA+ASC were statistically similar to the control. Inclusion of phage in treatments gradually decreased STEC loads when combined with ASC, PAA, and UV. Phage and UV combination led to the lowest STEC load (1.46 log reduction). Contrast analysis showed that treatments with phage significantly decreased STEC loads when compared to other treatments (P < 0.0001) and control (P < 0.0001). STEC loads recovered from treatments without phage and control were statistically similar at P = 0.07.ConclusionIndividual or combined applications of MS phages on beef surface contaminated with STEC provided optimal antimicrobial effect under vacuum or aerobic conditions. Although organic acids and UV combinations did reduce STEC populations, treatments that included phage yielded the lowest STEC loads. Only phage interventions gave optimal reduction effects under vacuum conditions. Antimicrobial treatments based on individual phage cocktails and their combinations with ASC, UV, and PAA significantly reduce STEC when treating primals produced during HEP.

Efficiency of Commercial Bacteriophages on Stec O157:H7 Populations in Beef Kept Under Vacuum and Aerobic Conditions

ObjectivesHigh event period (HEP) is a specific time length when processing facilities experience an elevated rate of STEC contamination. STEC contamination during beef fabrication is assessed by sampling trim combos usually using N60, N60 plus, or CSD cloth methods. However, beef primals produced during high event periods can also be affected and must be assessed. A common industry practice consists in reworking primals by removing them from vacuum sealed bags, treating with antimicrobials, repackaging, and then test for STEC. In this study, we evaluated the efficiency of bacteriophage and organic acid applications on contaminated beef kept under vacuum and aerobic conditions.Materials and MethodsAntimicrobials used in this study included: PhageGuard E (PGE, 108 PFU/ml, Bacteriophage solution from Micreos Food Safety BV), peroxyacetic acid (PAA, 400 PPM), and lactic acid (LA, 4.5% at 50°C). STEC O157:H7 strains included: ATCC® 35150 (stx1 and stx2 positive), ATCC® 43895 (stx1 and stx2 positive), ATCC® 43894 (stx1 and stx2 positive), and Micreos 128. Bacteriophage killing efficiency was determined for individual strains in vitro. Fresh rose meat (Cutaneous trunci) was cut into 100 cm2 and stored at 7°C. Meat samples (n = 160, 5 reps, 2 experimental units per rep) were randomly assigned to a 4×2×2 factorial whereas fixed effects were antimicrobial treatment (Control, PGE, PAA, and LA), packaging (V- vacuum and NV– aerobic), and lysing time (30 min and 6 h). Samples were inoculated with 500 ML of a STEC cocktail containing all 4 strains and after 30 min at 7°C under vacuum or wrapped in permeable film, samples were treated with 500 μL PGE, sterile buffered peptone water (BPW, Control), LA, or PAA. Samples were then re-vacuumed or re-wrapped with oxygen permeable film and kept either for 30 min or 6h at 7°C. After refrigeration, samples were swabbed and homogenized in 1mL of BPW. The swab content was serially diluted and spread-plated onto LB agar plates for bacterial enumeration. Data were analyzed using SAS as a completely randomized design.ResultsIn vitro killing efficiency was 98.3%, 96.7%, 97.2%, and 98.2% for Micreos 128, ATCC® 43894, ATCC® 43895, and ATCC® 35150 strains, respectively. When analyzing the effects of antimicrobials, packaging, and lysing time, a three-way interaction was observed (P = 0.035). Under aerobic conditions for 30 min, PGE reduced STEC in beef by approximately 1.4 log CFU/cm2 whereas organic acids reduced by 0.5 log. Similar results were observed when samples were kept for 6 h. Under vacuum conditions for 30 min, PGE significantly reduced STEC by 1 log, whereas no significant effects were observed when treating beef with PAA and LA. Under vacuum conditions for 6h, PGE significantly reduced STEC loads by 1.4 log, whereas LA reduced by 0.6 log and no differences were observed between control and PAA treatments.ConclusionBacteriophage applications on beef contaminated with STEC yielded the lowest counts when compared to PAA and LA. Although organic acids led to a significant decrease of STEC loads in beef kept under aerobic conditions, bacteriophage application led to the lowest counts. Similar to reworking and testing primals produced during a HEP, while under vacuum conditions, bacteriophage significantly reduced STEC loads whereas no or minimal effects of organic acids were observed.

The pathogenicity of rmpA or aerobactin-positive Klebsiella pneumoniae in infected mice

Objectives To investigate the pathogenicity of Klebsiella pneumoniae (KPN) possessing rmpA or the aerobactin gene in infected mice. Methods BALB/c mice were divided into four groups (n = 10 per group) and infected with: string test-positive and rmpA-positive or aerobactin-positive KPN (group 1), string test-negative but rmpA-positive KPN (group 2), string test-negative but aerobactin-positive KPN (group 3), or string test- and rmpA/aerobactin-negative KPN (group 4). Mouse survival time was compared among groups, and the infection of livers, spleens, lungs, and kidneys and KPN growth were assessed in infected mice. Results Compared with the negative group (group 4), the survival rates of mice infected with rmpA- or aerobactin-positive KPN (groups 1–3) were significantly lower, their multi-organ injuries were significantly more severe, and bacterial enumeration was significantly higher. Conclusions Despite being string test-negative, aerobactin- or rmpA-positive KPN still exhibit high virulence and anti-immune effect activity. Therefore, the combination of the string test and gene detection of aerobactin and rmpA will be helpful in screening hypervirulent KPN.