Quantification, Viability Assessment, and Visualization Strategies for <em>Acinetobacter</em> Biofilms

Acinetobacter causes nosocomial infections and its biofilm formation can contribute to the survival on dry surfaces such as hospital environments. Thus, ...

Korea Food Research Institute

Korea university of science and technology

Campylobacter jejuni is an important foodborne pathogen of humans and its primary reservoir is the gastrointestinal (GI) tract of chickens. Our previous studies demonstrated that phase variation to specific "successful alleles" at C. jejuni contingency loci Cj0045 (successful alleles carry 9G or 10G homopolymeric tracts) and Cj0170 (successful allele carries a 10G homopolymeric tract) in C. jejuni populations is strongly associated with colonization and enteritis in C57BL/6 IL-10 deficient mice. In the current study, we strengthened the association between locus Cj0170, Cj0045, and mouse colonization. We generated 8 independent strains derived from C. jejuni 11168 strain KanR4 that carried a Cj0170 gene disruption and these were all non motile. Two randomly chosen strains with the Cj0170 gene disruption (DM0170-2 and DM0170-6) were gavaged into mice. DM0170-2 and DM0170-6 failed to colonize mice while the control strain that carried a "successful"Cj0170 10G allele was motile and did colonize mice. In parallel studies, when we inoculated C. jejuni strain 33292 into mice, the "unsuccessful"Cj0045 11G allele experienced phase variation to "successful" 9G and 10G alleles in 2 independent experiments prior to d4 post inoculation in mice while the "successful" 9G allele in the control strain remained stable through d21 post inoculation or shifted to other successful alleles. These data confirm that locus Cj0170 regulates motility in C. jejuni strain KanR4 and is a virulence factor in the mouse model. The data also support a possible role of locus Cj0045 as a virulence factor in strain 33292 in infection of mice.

A "successful allele" at Campylobacter jejuni contingency locus Cj0170 regulates motility; "successful alleles" at locus Cj0045 are strongly associated with mouse colonization.

Air and nitrogen gas are commonly used feed gases for plasma generation and are economically useful in industrial applications. The two gases were compared in dielectric barrier discharge plasma for the inactivation of Campylobacter jejuni on an agar surface. Plasma treatment with nitrogen gas for 20 s did not yield any reduction (p>0.05) in viable cell count. However, a 0.8-log reduction (p<0.05) in colony-forming units (CFU) occurred when the nitrogen gas was supplemented with 2% (vol/vol) air. The use of air only, air supplemented with 2% (vol/vol) nitrogen, or oxygen only further decreased the viable cell counts by 0.7-1.7-log CFU (p<0.05). These results suggest that oxygen in plasma generation is critically important for the increased inactivation effect. Scanning electron microscopy analysis showed much cell debris including fragmented flagella in the sample exposed to air plasma, while no cell debris was found in the sample exposed to nitrogen plasma. In transmission electron microscopy analysis, many C. jejuni cells exposed to air plasma had truncated flagella with sharp bends, while the cells exposed to nitrogen plasma were normal, strongly suggesting that the air plasma can reduce the virulence of C. jejuni. A BacLight assay showed that air plasma damaged the cellular membrane (p<0.05), whereas nitrogen plasma did not after 5- or 20-s treatment. The damage to the membrane was consistent with the reduced viable cell count. Based on confocal microscopic analysis, the similar results were found by visualizing the fluorescent-dye-stained cells. In addition, the prolonged nitrogen plasma for 2 min also damaged many cellular membranes. This study shows that air, especially oxygen, is more effective and destructive than nitrogen and provides evidence that membrane damage may be a major mechanism for the inactivation of C. jejuni exposed to plasma.

Inactivation of Campylobacter jejuni with dielectric barrier discharge plasma using air and nitrogen gases.

The complex roles of flagella in the pathogenesis of Campylobacter jejuni, a major cause of worldwide foodborne diarrheal disease, are important. Compared with the wild-type, an insertional mutation of the flgA gene (cj0769c) demonstrated significant decrease in the biofilm formation of C. jejuni NCTC11168 on major food contact surfaces, such as polystyrene, stainless steel, and borosilicate glass. The flgA mutant was completely devoid of flagella and non-motile whereas the wild-type displayed the full-length flagella and motility. In addition, the biofilm formation of the wild-type was inversely dependent on the viscosity of the media. These results support that flagellar-mediated motility plays a significant role in the biofilm formation of C. jejuni NCTC11168. Moreover, our adhesion assay suggests that it plays an important role during biofilm maturation after initial attachment. Furthermore, C. jejuni NCTC11168 wild-type formed biofilm with a net-like structure of extracellular fiber-like material, but such a structure was significantly reduced in the biofilm of the flgA mutant. It supports that the extracellular fiber-like material may play a significant role in the biofilm formation of C. jejuni. This study demonstrated that flgA is essential for flagellar biosynthesis and motility, and plays a significant role in the biofilm formation of C. jejuni NCTC11168.

Role of flgA for Flagellar Biosynthesis and Biofilm Formation of Campylobacter jejuni NCTC11168.

 and  are important foodborne pathogenic bacteria, particularly in poultry meat. In this study, the presence of extracellular DNase activity was investigated for biofilm-deficient  strains versus biofilm-forming  strains isolated from chickens, to understand the relationship between extracellular DNase activity and biofilm formation. A biofilm-forming reference strain,  NCTC11168, was co-incubated with biofilm non-forming strains isolated from raw chickens or their supernatants. The biofilm non-forming strains or supernatants significantly prohibited the biofilm formation of  NCTC11168. In addition, the strains degraded pre-formed biofilms of  NCTC11168. Degradation of  NCTC11168 biofilm was confirmed after treatment with the supernatant of the biofilm non-forming strain 2-1 by confocal laser scanning microscopy. Quantitative analysis of the biofilm matrix revealed reduction of extracellular DNA (16%) and proteins (8.7%) after treatment. Whereas the biofilm-forming strains  Y23-5 and  34-3 isolated from raw chickens and the  NCTC11168 reference strain showed no extracellular DNase activity against their own genomic DNA, most biofilm non-forming strains tested, including  2-1,  34-1, and  63-1, exhibited obvious extracellular DNase activities against their own or 11168 genomic DNA, except for one biofilm non-former,  22-1. Our results suggest that extracellular DNase activity is a common feature suppressing biofilm formation among biofilm non-forming  or  strains of chicken origin.

Inverse Correlation between Extracellular DNase Activity and Biofilm Formation among Chicken-Derived  Strains.

This study investigated the effects of enzyme application on biofilms of bacterial isolates from a cafeteria kitchen and foodborne pathogens and the susceptibility of Salmonella biofilms to proteinase K combined with chlorine treatment. For four isolates from a cafeteria kitchen ( Acinetobacter, Enterobacter, and Kocuria) and six strains of foodborne pathogens ( Salmonella enterica, Staphylococcus aureus, and Vibrio parahaemolyticus), the inhibitory effect of enzymes on biofilm formation at 25°C for 24 h or the degradative efficacy of enzymes on 24-h mature biofilm at 37°C for 1 h in tryptic soy broth (TSB) was examined in a polystyrene microtiter plate. The effect of enzymes was also evaluated on a subset of these strains in 20 times diluted TSB (1/20 TSB) at 25°C. The working concentrations of five enzymes were 1 U/100 μL for α-amylase, amyloglucosidase, cellulase, and DNase and 1 milli-Anson unit/100 μL for proteinase K. In addition, 24-h mature Salmonella Typhimurium biofilm on a stainless steel coupon was treated with proteinase K for 1 h at 25°C followed by 20 ppm of chlorine for 1 min at 25°C. The results showed that certain enzymes inhibited biofilm formation by the kitchen-originated bacteria; however, the enzymatic effect was diminished on the mature biofilms. Biofilm formation of V. parahaemolyticus was suppressed by all tested enzymes, whereas the mature biofilm was degraded by α-amylase, DNase I, and proteinase K. Proteinase K was effective in controlling Salmonella biofilms, whereas a strain-dependent variation was observed in S. aureus biofilms. In 1/20 TSB, Enterobacter cancerogenus and Kocuria varians were more susceptible to certain enzymes during biofilm formation than those in TSB, whereas the enzymatic effect was much decreased on 24-h mature biofilms, regardless of nutrient conditions. Furthermore, synergistic inactivation of Salmonella Typhimurium in biofilms was observed in the combined treatment of proteinase K followed by chlorine. Live/Dead assays also revealed a decrease in density and loss of membrane integrity in Salmonella Typhimurium biofilms exposed to the combined treatment. Therefore, certain enzymes can control biofilms of isolates residing in a cafeteria kitchen and foodborne pathogens. This study demonstrates the potential of enzymes for the sanitation of food processing environments and of proteinase K combined with chlorine to control Salmonella biofilms on food contact surfaces.

Enzymatic Inactivation of Pathogenic and Nonpathogenic Bacteria in Biofilms in Combination with Chlorine.

Escherichia coli O157:H7 is one of the most important pathogens worldwide. In this study, three different kinds of enzymes, DNase I, proteinase K and cellulase were evaluated for inhibitory or degrading activity against E. coli O157:H7 biofilm by targeting extracellular DNA, proteins, and cellulose, respectively. The cell number of biofilms formed under proteinase K resulted in a 2.43 log CFU/cm reduction with an additional synergistic 3.72 log CFU/cm reduction after NaClO post-treatment, while no significant reduction occurred with NaClO treatment alone. It suggests that protein degradation could be a good way to control the biofilm effectively. In preformed biofilms, all enzymes showed a significant reduction of 16.4-36.7% in biofilm matrix in 10-fold diluted media (p < 0.05). The sequential treatment with proteinase K, cellulase, and NaClO showed a significantly higher synergistic inactivation of 2.83 log CFU/cm compared to 1.58 log CFU/cm in the sequence of cellulase, proteinase K, and NaClO (p < 0.05). It suggests that the sequence of multiple enzymes can make a significant difference in the susceptibility of biofilms to NaClO. This study indicates that the combination of extracellular polymeric substance-degrading enzymes with NaClO could be useful for the efficient control of E. coli O157:H7 biofilms.

Bio-enzymes for inhibition and elimination of Escherichia coli O157:H7 biofilm and their synergistic effect with sodium hypochlorite.

Bacteria can survive and persist in food processing environments by attachment and biofilm formation and transfer to food products, causing serious foodborne illness. In this study, we investigated natural substances that belong to the family  to determine whether they have potential anti-attachment activities against  O157:H7. The inhibition of biofilm formation was evaluated by crystal violet and resazurin assays at different stages of biofilm formation (initial attachment, biofilm formation, and after biofilm development) of  O157:H7. The sessile cells were reduced to a range of 13.8-31.3% by young radish, radish, radish sprout, red cabbage, and kale extracts, and the viability was reduced to between 5.83 and 51.5%. The radical scavenging activities and the presence of polyphenolic compounds were compared. The presence of phenolic compounds such as gallic acid, caffeic acid, and phenylethyl ITC in the  family verified the potential use as a natural anti-biofilm substituent against  O157:H7.

Anti-attachment, anti-biofilm, and antioxidant properties of  extracts on  O157:H7.

Insufficient and ineffective cleaning practices can cause food residues to remain in kitchen and can facilitate bacterial attachment and persistence by protective films. The present study investigated the survival of five major foodborne pathogens on stainless steel coupons, in the presence of cooked rice, whole eggs, and soymilk. Foodborne pathogens showed different survival rates by desiccation and disinfection depending on food residues. Overall, the pathogens showed stronger survival than the control at 0.13-3.97 log CFU/coupon with 5% residues, and at 0.75-5.29 log CFU/coupon with 50% residues.  was not affected by the food residue with showing the least difference in concentration, while  O157:H7 showed the most significant increase by food residue. The cells with cooked rice were observed using FE-SEM, and demonstrated bacterial binding or embedment. All results suggest that food safety can be practically ensured by food residue types and appropriate cleaning and disinfectants.

Survival of foodborne pathogens on stainless steel soiled with different food residues.

Bacteria can form biofilms, complex microbial communities protected from environmental stress, on food contact surfaces. Brassicaceae plant has been shown to contain bioactive compounds with antimicrobial activities. The objective of this study was to evaluate the synergistic effects of Brassicaceae species and proteinase K against E. coli O157:H7 biofilm. We determined the minimum biofilm inhibitory concentration, the fractional inhibitory concentration indexes, and the synergistic inhibitory effect of Raphanus sativus var. longipinnatus, R. sativus, and Brassica oleracea var. acephala extracts with proteinase K on E. coli O157:H7. The biofilm showed a 49% reduction with 2 mg/mL R. sativus. The combination of proteinase K 25 µg/mL significantly increased the effect of 2 mg/mL R. sativus var. longipinnatus and the combined treatment yielded up to 2.68 log reduction on stainless steel coupons. The results showed that the combination of R. sativus var. longipinnatus extract and proteinase K could serve as an anti-biofilm agent with synergistic effects for inhibiting E. coli O157:H7 biofilm on stainless steel surfaces.

Synergistic anti-biofilm effects of Brassicaceae plant extracts in combination with proteinase K against Escherichia coli O157:H7.

Bacterial contamination of food-contact surfaces can be a potential risk factor for food quality and safety. To evaluate the spatial and temporal variations of the potential cross-contamination routes, we conducted a biogeographical assessment of bacteria in a foodservice facility based on the diversity of microflora on each surface. To this end, we performed high-throughput amplicon sequencing of 13 food-contact and non-food contact surfaces in a foodservice facility throughout a year. The results showed that , , , , , , , , and  were the dominant genera found on the kitchen surfaces of the foodservice facility. Depending on the season, changes in  ratios were observed, and the fan becomes the main source of outdoor air contamination. The microbial flow associated with spoilage was also observed throughout food preparation. Taken together, our results would be a powerful reference to hygiene managers for improvement of food processes.

Metagenomic Analysis of Microbial Composition Revealed Cross-Contamination Pathway of Bacteria at a Foodservice Facility.

Bacillus cereus is a foodborne pathogen and can form biofilms on food contact surfaces, which causes food hygiene problems. While it is necessary to understand strain-dependent variation to effectively control these biofilms, strain-to-strain variation in the structure of B. cereus biofilms is poorly understood. In this study, B. cereus strains from tatsoi (BC4, BC10, and BC72) and the ATCC 10987 reference strain were incubated at 30°C to form biofilms in the presence of the extracellular matrix-degrading enzymes DNase I, proteinase K, dispase II, cellulase, amyloglucosidase, and α-amylase to assess the susceptibility to these enzymes. The four strains exhibited four different patterns in terms of biofilm susceptibility to the enzymes as well as morphology of surface-attached biofilms or suspended cell aggregates. DNase I inhibited the biofilm formation of strains ATCC 10987 and BC4 but not of strains BC10 and BC72. This result suggests that some strains may not have extracellular DNA, or their extracellular DNA may be protected in their biofilms. In addition, the strains exhibited different patterns of susceptibility to protein- and carbohydrate-degrading enzymes. While other strains were resistant, strains ATCC 10987 and BC4 were susceptible to cellulase, suggesting that cellulose or its similar polysaccharides may exist and play an essential role in their biofilm formation. Our compositional and imaging analyses of strains ATCC 10987 and BC4 suggested that the physicochemical properties of their biofilms are distinct, as calculated by the carbohydrate to protein ratio. Taken together, our study suggests that the extracellular matrix of B. cereus biofilms may be highly diverse and provides insight into the diverse mechanisms of biofilm formation among B. cereus strains.

Strain variation in Bacillus cereus biofilms and their susceptibility to extracellular matrix-degrading enzymes.

Foodborne pathogenic bacteria in multi-species biofilms in food manufacturing facilities have been suspected to be the cause of cross-contamination leading to foodborne illness. We studied if cafeteria kitchen-associated bacterial isolates can have any protective effect on E. coli O157:H7 in biofilm against extracellular polymeric substances (EPS)-degrading enzymes and sodium hypochlorite. We investigated multi-species biofilm-forming ability and the efficacy of EPS-degrading enzymes using crystal violet assay. The susceptibility of E. coli O157:H7 to sodium hypochlorite (NaClO) was evaluated using propidium monoazide combined with quantitative PCR (PMA-qPCR). Then, a combined treatment with enzymes followed by NaClO was also tested. Most cafeteria kitchen isolates of Acinetobacter and Bacillus were able to form biofilms. Several of them showed a protective effect on E. coli O157:H7 against NaClO after forming multi-species biofilms, particularly in Acinetobacter. This protective effect on E. coli O157:H7 was also noticed after the enzyme or the combined treatment with NaClO. Our results give us an insight into the protective role of food-associated environmental bacteria for E. coli O157:H7 in biofilms against common sanitizers and warrant further study to develop effective control methods. Our study also highlights the importance of preventing contamination or biofilm formation by environmental microorganisms, eventually reducing foodborne illness.

Protective role of Acinetobacter and Bacillus for Escherichia coli O157:H7 in biofilms against sodium hypochlorite and extracellular matrix-degrading enzymes.

Joo-Sung Kim

Korea Food Research Institute

Quantification, Viability Assessment, and Visualization Strategies for Acinetobacter Biofilms