Pressure- vs. heat-induced bacterial stress in cooked poultry sausages: a preliminary study

Vacuum-packaged poultry cooked sausages were pressure-treated at 500 MPa by combinations of time (5-45 min) and temperature (2-80 degrees C) and later stored at 6-8 degrees C for 12 we. Mesophile and psychrotrophe counts were determined 1 d, 3, 6, 9 and 12 we after treatment and compared with those of cooked sausages pasteurized at 80-85 degrees C for 40 min. Both pressure and heat treatments offer great possibilities for preservation. Sausages pressurized at 65 degrees C for 15 min showed mesophile numbers below 2 log cfu g(-1) throughout the chill storage. Pressurization, unlike heat treatment, causes a reversible bacterial stress. Thus, injured cells recovered during storage and, at 6 and 12 we, after a temperature abuse (room temperature for approx. 24 h), counts increased up to 6.5 - 7.5 log units. Psychrotrophes were more sensitive to both treatments; no growth was detected the day after (a lethality of more than 4 log units).     

Biocontrol of Listeria monocytogenes on fresh-cut produce by treatment with lytic bacteriophages and a bacteriocin

The fresh-cut produce industry has been the fastest-growing portion of the food retail market during the past 10 years, providing consumers with convenient and nutritious food. However, fresh-cut fruits and vegetables raise food safety concerns, because exposed tissue may be colonized more easily by pathogenic bacteria than intact produce. This is due to the higher availability of nutrients on cut surfaces and the greater potential for contamination because of the increased amount of handling. We found that applied Listeria monocytogenes populations survived and increased only slightly on fresh-cut Red Delicious apples stored at 10 degrees C but increased significantly on fresh-cut honeydew melons stored at 10 degrees C over 7 days. In addition, we examined the effect of lytic, L. monocytogenes-specific phages via two phage application methods, spraying and pipetting, on L. monocytogenes populations in artificially contaminated fresh-cut melons and apples. The phage mixture reduced L. monocytogenes populations by 2.0 to 4.6 log units over the control on honeydew melons. On apples, the reduction was below 0.4 log units. In combination with nisin (a bacteriocin), the phage mixture reduced L. monocytogenes populations by up to 5.7 log units on honeydew melon slices and by up to 2.3 log units on apple slices compared to the control. Nisin alone reduced L. monocytogenes populations by up to 3.2 log units on honeydew melon slices and by up to 2.0 log units on apple slices compared to the control. The phage titer was stable on melon slices, but declined rapidly on apple slices. The spray application of the phage and phage plus nisin reduced the bacterial numbers at least as much as the pipette application. The effectiveness of the phage treatment also depended on the initial concentration of L. monocytogenes.     

Sensitization of Listeria monocytogenes to Low pH, Organic Acids, and Osmotic Stress by Ethanol

The killing of Listeria monocytogenes following exposure to low pH, organic acids, and osmotic stress was enhanced by the addition of 5% (vol/vol) ethanol. At pH 3, for example, the presence of this agent stimulated killing by more than 3 log units in 40 min of exposure. The rate of cell death at pH 3.0 was dependent on the concentration of ethanol. Thus, while the presence 10% (vol/vol) ethanol at pH 3.0 stimulated killing by more than 3 log units in just 5 min, addition of 1.25% (vol/vol) ethanol resulted in less than 1 log unit of killing in 10 min. The ability of 5% (vol/vol) ethanol to stimulate killing at low pH and at elevated osmolarity was also dependent on the amplitude of the imposed stress, and an increase in the pH from 3.0 to 4.0 or a decrease in the sodium chloride concentration from 25 to 2.5% led to a marked reduction in the effectiveness of 5% (vol/vol) ethanol as an augmentative agent. Combinations of organic acids, low pH, and ethanol proved to be particularly effective bactericidal treatments; the most potent combination was pH 3.0, 50 mM formate, and 5 % (vol/vol) ethanol, which resulted in 5 log units of killing in just 4 min. Ethanol-enhanced killing correlated with damage to the bacterial cytoplasmic membrane.     

Reduction of experimental Salmonella and Campylobacter contamination of chicken skin by application of lytic bacteriophages

Lytic bacteriophages, applied to chicken skin that had been experimentally contaminated with Salmonella enterica serovar Enteritidis or Campylobacter jejuni at a multiplicity of infection (MOI) of 1, increased in titer and reduced the pathogen numbers by less than 1 log(10) unit. Phages applied at a MOI of 100 to 1,000 rapidly reduced the recoverable bacterial numbers by up to 2 log(10) units over 48 h. When the level of Salmonella contamination was low (< log(10) 2 per unit area of skin) and the MOI was 10(5), no organisms were recovered. By increasing the number of phage particles applied (i.e., MOI of 10(7)), it was also possible to eliminate other Salmonella strains that showed high levels of resistance because of restriction but to which the phages were able to attach.     

A laboratory hot tub model for disinfectant efficacy evaluation

This paper describes a novel laboratory hot tub (LHT) apparatus and associated standard operating procedure (SOP) designed to reproduce the key biological, chemical, and engineering parameters associated with recreational and therapeutic hot tubs. Efficacy, as measured quantitatively by log reduction values, was determined against both biofilm and planktonic bacteria. When the LHT was run according to the SOP, with no antimicrobial treatment, a consistent level of bacterial contamination occurred. The means of log10 viable cell densities (+/- the repeatability standard deviation of log densities) were 7.2 (+/-0.31) for the bulk water (density in units of cfu ml-1), 5.3 (+/-0.56) for the coupons (density in units of cfu cm-2), and 6.6 (+/-0.50) for the filters (density in units of cfu cm-2). When control and chlorine treated LHTs were run in parallel, the log reduction increased significantly with chlorine concentration for samples of planktonic bacteria in the bulk water (p=0.016), biofilm bacteria on the coupons (p=0.09) and biofilm bacteria on the filter (p=0.005), indicating that the method was sensitive to chlorine concentration. The method also displayed sensitivity by differentiating between chlorine and bromine treatments; in every case, chlorine produced a greater log reduction than did the same concentration of bromine. The model and SOP were shown to be rugged with respect to slight changes in fluid mixing intensity, water chemistry (saturation index), inoculum size, and organic loading. The LHT and associated SOP provide a reliable second tier in a three-tiered testing process, in which the first tier is a suspension test and the final tier is a field test.     

Comparative survival of free shiga toxin 2-encoding phages and Escherichia coli strains outside the gut

The behavior outside the gut of seeded Escherichia coli O157:H7, naturally occurring E. coli, somatic coliphages, bacteriophages infecting O157:H7, and Shiga toxin 2 (Stx2)-encoding bacteriophages was studied to determine whether the last persist in the environment more successfully than their host bacteria. The ratios between the numbers of E. coli and those of the different bacteriophages were clearly lower in river water than in sewage of the area, whereas the ratios between the numbers of the different phages were similar. In addition, the numbers of bacteria decreased between 2 and 3 log units in in situ survival experiments performed in river water, whereas the numbers of phages decreased between 1 and 2 log units. Chlorination and pasteurization treatments that reduced by approximately 4 log units the numbers of bacteria reduced by less than 1 log unit the numbers of bacteriophages. Thus, it can be concluded that Stx2-encoding phages persist longer than their host bacteria in the water environment and are more resistant than their host bacteria to chlorination and heat treatment.     

Biocontrol of Listeria monocytogenes on Fresh-Cut Produce by Treatment with Lytic Bacteriophages and a Bacteriocin

The fresh-cut produce industry has been the fastest-growing portion of the food retail market during the past 10 years, providing consumers with convenient and nutritious food. However, fresh-cut fruits and vegetables raise food safety concerns, because exposed tissue may be colonized more easily by pathogenic bacteria than intact produce. This is due to the higher availability of nutrients on cut surfaces and the greater potential for contamination because of the increased amount of handling. We found that applied Listeria monocytogenes populations survived and increased only slightly on fresh-cut Red Delicious apples stored at 10°C but increased significantly on fresh-cut honeydew melons stored at 10°C over 7 days. In addition, we examined the effect of lytic, L. monocytogenes-specific phages via two phage application methods, spraying and pipetting, on L. monocytogenes populations in artificially contaminated fresh-cut melons and apples. The phage mixture reduced L. monocytogenes populations by 2.0 to 4.6 log units over the control on honeydew melons. On apples, the reduction was below 0.4 log units. In combination with nisin (a bacteriocin), the phage mixture reduced L. monocytogenes populations by up to 5.7 log units on honeydew melon slices and by up to 2.3 log units on apple slices compared to the control. Nisin alone reduced L. monocytogenes populations by up to 3.2 log units on honeydew melon slices and by up to 2.0 log units on apple slices compared to the control. The phage titer was stable on melon slices, but declined rapidly on apple slices. The spray application of the phage and phage plus nisin reduced the bacterial numbers at least as much as the pipette application. The effectiveness of the phage treatment also depended on the initial concentration of L. monocytogenes.     

Comparative Survival of Free Shiga Toxin 2-Encoding Phages and Escherichia coli Strains outside the Gut

The behavior outside the gut of seeded Escherichia coli O157:H7, naturally occurring E. coli, somatic coliphages, bacteriophages infecting O157:H7, and Shiga toxin 2 (Stx2)-encoding bacteriophages was studied to determine whether the last persist in the environment more successfully than their host bacteria. The ratios between the numbers of E. coli and those of the different bacteriophages were clearly lower in river water than in sewage of the area, whereas the ratios between the numbers of the different phages were similar. In addition, the numbers of bacteria decreased between 2 and 3 log units in in situ survival experiments performed in river water, whereas the numbers of phages decreased between 1 and 2 log units. Chlorination and pasteurization treatments that reduced by approximately 4 log units the numbers of bacteria reduced by less than 1 log unit the numbers of bacteriophages. Thus, it can be concluded that Stx2-encoding phages persist longer than their host bacteria in the water environment and are more resistant than their host bacteria to chlorination and heat treatment.     

Effectiveness of phages in the decontamination of Listeria monocytogenes adhered to clean stainless steel, stainless steel coated with fish protein, and as a biofilm

Listeria monocytogenes is a food-borne pathogen which causes listeriosis and is difficult to eradicate from seafood processing environments; therefore, more effective control methods need to be developed. This study investigated the effectiveness of three bacteriophages (LiMN4L, LiMN4p and LiMN17), individually or as a three-phage cocktail at ≈9 log₁₀ PFU/ml, in the lysis of three seafood-borne L. monocytogenes strains (19CO9, 19DO3 and 19EO3) adhered to a fish broth layer on stainless steel coupon (FBSSC) and clean stainless steel coupon (SSC), in 7-day biofilm, and dislodged biofilm cells at 15 ± 1 °C. Single phage treatments (LiMN4L, LiMN4p or LiMN17) decreased bacterial cells adhered to FBSSC and SSC by ≈3–4.5 log units. Phage cocktail reduced the cells on both surfaces (≈3.8–4.5 and 4.6–5.4 log₁₀ CFU/cm², respectively), to less than detectable levels after ≈75 min (detection limit = 0.9 log₁₀ CFU/cm²). The phage cocktail at ≈5.8, 6.5 and 7.5 log₁₀ PFU/cm² eliminated Listeria contamination (≈1.5–1.7 log₁₀ CFU/cm²) on SSC in ≈15 min. One-hour phage treatments (LiMN4p, LiMN4L and cocktail) in three consecutive applications resulted in a decrease of 7-day L. monocytogenes biofilms (≈4 log₁₀ CFU/cm²) by ≈2–3 log units. Single phage treatments reduced dislodged biofilm cells of each L. monocytogenes strain by ≈5 log₁₀ CFU/ml in 1 h. The three phages were effective in controlling L. monocytogenes on stainless steel either clean or soiled with fish proteins which is likely to occur in seafood processing environments. Phages were more effective on biofilm cells dislodged from the surface compared with undisturbed biofilm cells. Therefore, for short-term phage treatments of biofilm it should be considered that some disruption of the biofilm cells from the surface prior to phage application will be required.     

Chlorination of indicator bacteria and viruses in primary sewage effluent

Wastewater disinfection is used in many countries for reducing fecal coliform levels in effluents. Disinfection is therefore frequently used to improve recreational bathing waters which do not comply with microbiological standards. It is unknown whether human enteric viruses (which are responsible for waterborne disease) are simultaneously inactivated alongside fecal coliforms. This laboratory study focused on the chlorination of primary treated effluent with three doses (8, 16, and 30 mg/liter) of free chlorine as sodium hypochlorite. Seeding experiments showed that inactivation (>5 log(10) units) of Escherichia coli and Enterococcus faecalis was rapid and complete but that there was poor inactivation (0.2 to 1.0 log(10) unit) of F(+)-specific RNA (FRNA) bacteriophage (MS2) (a potential virus indicator) at all three doses. However, seeded poliovirus was significantly more susceptible (2.8 log(10) units) to inactivation by chlorine than was the FRNA bacteriophage. To ensure that these results were not artifacts of the seeding process, comparisons were made between inactivation rates of laboratory-seeded organisms in sterilized sewage and inactivation rates of organisms occurring naturally in sewage. Multifactorial analysis of variance showed that there was no significant difference (P > 0.05) between the inactivation rates for seeded and naturally occurring FRNA bacteriophage. However, laboratory-grown poliovirus was inactivated much more rapidly than were naturally occurring, indigenous enteroviruses (P < 0.001). This may reflect differences in the way indigenous virus is presented to the disinfectant. Inactivation rates for indigenous enteroviruses were quite similar to those seen for FRNA bacteriophage at lower doses of chlorine. These results have significance for the effectiveness of chlorination as a sewage treatment process, particularly where virus contamination is of concern, and suggest that FRNA bacteriophage would be an appropriate indicator of such viral inactivation under field conditions.     

Resistance of Faecal Coliforms and Enterococci Populations in Sludge and Biosolids to Different Hygienisation Treatments

The composition of the most abundant facultative anaerobic bacteria populations [faecal coliforms (FC) and enterococci (ENT)] in sludge can be modified after different treatments. These involve the disposal or reuse of sludge and include: anaerobic digesters, incineration, composting, pasteurization and lime treatments. In this study, three treatment types (mesophilic anaerobic digestion, composting and pasteurization) were compared in terms of their ability to reduce both bacterial populations. The diversity and any changes in composition of main phenotypic groups for both populations were also analyzed. Mesophilic anaerobic digestion (MAD) was carried out at 35°C for 20 days. Digested sludge was then dehydrated by centrifugation at 2,500 rpm. Composting (COM) was performed at 55°C with windrow phases. Pasteurization was assayed at 60°C for 90 min (P60), at 80°C for 60 min (P80). A 1–1.5 log unit reduction was observed for FC, and 1 log unit reduction was noted for ENT by MAD treatment. In composting, this reduction proved higher for FC than for ENT (6 log and 3–4 log units, respectively). Optimal pasteurization was obtained at 80°C for 60 min, resulting in a 5 log unit reduction for FC and a 2 log unit reduction for ENT. High diversity indices (Di) for both bacterial populations were detected both before and after implementation of the different treatments. Analyses of the population’s similarity provided that FC were diverse both before and after COM, P60 and P80 treatments. However, no differences were observed on the composition of ENT populations after the different treatments assayed.     

Pulsed-electric field treatment enhances the bactericidal action of nisin against Bacillus cereus

Vegetative cells of Bacillus cereus were subjected to low doses of nisin (0.06 microg/ml) and mild pulsed-electric field treatment (16.7 kV/cm, 50 pulses each of 2-micros duration). Combining both treatments resulted in a reduction of 1.8 log units more than the sum of the reductions obtained with the single treatments, indicating synergy.     

The hygienic efficiency of conventional and inverted lamb dressing systems

R.G. BELL AND S.C. HATHAWAY. 1996. Aerobic plate counts (APC 37°C and APC 25°C) and Escherichia coli enumerations (Petrifilm) were used to determine sources of bacterial contamination during sheep dressing, determine the hygienic efficacy of hand wash and knife 'sterilization'procedures and compare the hygiene efficiency of conventional and inverted sheep dressing systems. The major slaughterline sources of microbial contamination were: fleece > workers' hands > faecal pellets > knife blades. Aerobic plate counts (APC 37°C) exceeding log 4.4 cfu cm^-2 were considered indicative of direct fleece contact, whereas E. coli numbers exceeding log 3.3 cfu cm^-2 were considered indicative of direct faecal contact. A 44°C water hand rinse removed 90% of the microbial contamination from workers' hands, but rinsed hands, particularly those contacting the fleece, still carried a microbial population exceeding log 4.0 cfu cm^-2. A 44°C rinse followed by an 82°C water dip reduced the contamination on knife blades to less than log 3.0 cfu cm^-2. Inverted dressing systems produced carcasses with a lower contamination level than conventional systems. With both systems little increase in contamination occurred after pelt removal. The areas of highest contamination were the forequarter region with inverted dressing and the hindquarter with conventional dressing. In both cases these regions are the sites where cuts are made through the skin. With both systems contamination around these cuts was entirely consistent with direct fleece contact resulting from 'rollback'.     

Sensitization of Listeria monocytogenes to low pH, organic acids, and osmotic stress by ethanol

The killing of Listeria monocytogenes following exposure to low pH, organic acids, and osmotic stress was enhanced by the addition of 5% (vol/vol) ethanol. At pH 3, for example, the presence of this agent stimulated killing by more than 3 log units in 40 min of exposure. The rate of cell death at pH 3.0 was dependent on the concentration of ethanol. Thus, while the presence 10% (vol/vol) ethanol at pH 3.0 stimulated killing by more than 3 log units in just 5 min, addition of 1.25% (vol/vol) ethanol resulted in less than 1 log unit of killing in 10 min. The ability of 5% (vol/vol) ethanol to stimulate killing at low pH and at elevated osmolarity was also dependent on the amplitude of the imposed stress, and an increase in the pH from 3.0 to 4.0 or a decrease in the sodium chloride concentration from 25 to 2.5% led to a marked reduction in the effectiveness of 5% (vol/vol) ethanol as an augmentative agent. Combinations of organic acids, low pH, and ethanol proved to be particularly effective bactericidal treatments; the most potent combination was pH 3.0, 50 mM formate, and 5 % (vol/vol) ethanol, which resulted in 5 log units of killing in just 4 min. Ethanol-enhanced killing correlated with damage to the bacterial cytoplasmic membrane.     

A rapid method for detecting bacterial contamination in the presence of Penicillium and Streptomyces antibiotic fermentations

Contamination in antibiotic fermentation processes results in major economic and process problems. The detection and elimination of contamination is a continual objective for fermentation companies. While efforts continue to eliminate contamination by improving equipment and sterile techniques, it is still imperative to have a rapid method for detecting contamination in laboratory-stage inoculum and seed tanks. This article describes the successful studies leading to the adoption of the BACTEC, an automatic bacterial detection system, as a supplemental detection technique. The BACTEC system detects contamination by incubating samples with a selected(14)C-labeled substrate or substrates. The resulting metabolism of substrate produces (14)C-labeled CO(2) which is then quantified and expressed as a growth index, permitting detection of contamination more rapidly at a much earlier time than is possible with conventional detection techniques that involve Phenol red dextrose broth, streak plates, and microscopic examination techniques.     

Virus elimination during the purification of monoclonal antibodies by column chromatography and additional steps

The theoretical potential for virus transmission by monoclonal antibody based therapeutic products has led to the inclusion of appropriate virus reduction steps. In this study, virus elimination by the chromatographic steps used during the purification process for two (IgG‐1 & ?3) monoclonal antibodies (MAbs) have been investigated. Both the Protein G (>7log) and ion‐exchange (5 log) chromatography steps were very effective for eliminating both enveloped and non‐enveloped viruses over the life‐time of the chromatographic gel. However, the contribution made by the final gel filtration step was more limited, i.e., 3 log. Because these chromatographic columns were recycled between uses, the effectiveness of the column sanitization procedures (guanidinium chloride for protein G or NaOH for ion‐exchange) were tested. By evaluating standard column runs immediately after each virus spiked run, it was possible to directly confirm that there was no cross contamination with virus between column runs (guanidinium chloride or NaOH). To further ensure the virus safety of the product, two specific virus elimination steps have also been included in the process. A solvent/detergent step based on 1% triton X‐100 rapidly inactivating a range of enveloped viruses by >6 log inactivation within 1 min of a 60 min treatment time. Virus removal by virus filtration step was also confirmed to be effective for those viruses of about 50 nm or greater. In conclusion, the combination of these multiple steps ensures a high margin of virus safety for this purification process. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1341–1347, 2014     

Bacteriological examination of a modern animal house containing small laboratory animals.

Floors and other areas totalling 1800 m2, comprising conventional and specified-pathogen-free (SPF) units, were screened bacteriologically 6 times in a year. The contamination indices observed were lower within than outside the units, and lower in the SPF than in the conventional unit. Bacterial counts in rooms containing animals in the conventional and SPF units were very similar. In all of the areas investigated within the units, most of the samples revealed less than 2 colony forming units per cm2. In contrast, high degrees of bacteriological contamination were detected in the changing rooms after showering or washing before entry. Staphylococcus epidermidis was the dominant bacterial species isolated. The bacteriological spectrum did not vary between the areas surveyed.     

An acute osteomyelitis model in traumatized rat tibiae involving sand as a foreign body, thermal injury, and bimicrobial contamination

The multfactorial nature of bone injuries in modern warfare and emergency trauma patients warrants enhancement of existing models. To develop a more appropriate model, rat tibiae (n = 195) were mechanically injured, divided into 2 groups (with or without thermal injury), and contaminated with a range of Staphylococcus aureus (Cowan 1) inocula. In some experiments, S. aureus inocula also contained Escherichia coli or foreign bodies (sand or soil). The primary outcome measure was the amount of S. aureus remaining in the tibia (tibial bacterial load) 24 h after contamination, reported as log10 cfu/g bone. S. aureus showed ID50 and ID95 values of 72 and 977 cfu, respectively. Values were lower than seen previously by using S. aureus strain SMH. S. aureus tibial bacterial loads were higher in tibiae with mechanical and thermal injury (log10 4.15 +/- 0.27 cfu/g) versus mechanical injury alone (log10 3.1 +/- 0.47 cfu/g, P = 0.028). The addition of E. coli to the S. aureus inoculum had no effect on tibial bacterial loads (S. aureus only, log10 4.24 +/- 0.92 cfu/g; S. aureus + E. coli, log10 4.1 +/- 1.0 cfu/g, P = 0.74). Sand, added as a foreign body, increased tibial bacterial load. Combined mechanical and thermal trauma of the tibia is associated with increased S. aureus tibial bacterial loads, increasing the risk of acute osteomyelitis. Understanding the interplay of mechanical and thermal injuries, bimicrobial contamination, and foreign bodies may improve our understanding of traumatic bone injuries and the pathogenesis of osteomyelitis.     

Pulsed-Electric Field Treatment Enhances the Bactericidal Action of Nisin against Bacillus cereus

Vegetative cells of Bacillus cereus were subjected to low doses of nisin (0.06 μg/ml) and mild pulsed-electric field treatment (16.7 kV/cm, 50 pulses each of 2-μs duration). Combining both treatments resulted in a reduction of 1.8 log units more than the sum of the reductions obtained with the single treatments, indicating synergy.     

Quantitative analysis of bacterial aerosols in two different dental clinic environments.

Microbial aerosols are generated during dental treatments and may represent an important source of infection. This study was designed to quantify bacterial air contamination during dental treatments in both a closed dental operatory and a multichair dental clinic. Air was sampled by using a slit type of biological air sampler. Following air sampling, blood-supplemented Trypticase soy agar plates were incubated at 37 degrees C under anaerobic conditions for 7 days. The maximum levels of air contamination in the closed dental operatory were observed while dental treatments were being performed (four trials; 216 +/- 75 CFU/m3 for ultrasonic scaling treatments and 75 +/- 22 CFU/m3 for operative treatments). At 2 h after completion of the treatments, the bacterial counts were about the same as the pretreatment levels (12 to 14 CFU/m3). In the second part of the study, a multichair dental clinic was divided into four areas, and air contamination was monitored at each site. Three sites were located in active dental treatment areas, whereas no dental treatments were performed within an 11-m radius of the fourth site. At 3 h after the beginning of dental treatments, the highest bacterial counts were obtained in the three active dental treatment areas (76 to 114 CFU/m3). However, there was noticeable contamination in the inactive dental treatment area (42 CFU/m3). Thus, bacterial aerosols were able to spread into areas where there was no dental activity. My data show that dental treatments significantly increased the levels of bacterial air contamination in both a closed dental operatory and a multichair dental clinic.(ABSTRACT TRUNCATED AT 250 WORDS)