AEM Accepted Manuscript Posted Online 26 May 2017 Appl Environ Microbiol doi:10.1128/AEM.00628-17 Copyright © 2017 Marti et al This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license Biofilm Formation Potential of Heat Resistant Escherichia coli Dairy Isolates and Complete Genome of MDR Heat Resistant Strain FAM21845 Roger Martia, Michael Schmidb, Sandra Kullia, Kerstin Schneebergerb, Javorka Naskovaa, Susanne Knøchelc, Christian H Ahrensb, and Jörg Hummerjohanna# Agroscope, Division of Food Microbial Systems, Microbiological Safety of Foods of Animal Origin Group, Bern, Switzerlanda; Agroscope, Research Group Molecular 10 Diagnostics, Genomics and Bioinformatics & SIB Swiss Institute of Bioinformatics, 11 Wädenswil, Switzerlandb; Department of Food Science, University of Copenhagen, 12 Copenhagen, Denmarkc 13 14 Running Head: Biofilm formation of heat resistant E coli strains 15 16 # Address correspondence 17 joerg.hummerjohann@agroscope.admin.ch to J Hummerjohann, 18 19 Keywords: Biofilm, locus of heat resistance, LHR, Escherichia coli, persistence, 20 antimicrobial resistance, dairy Downloaded from http://aem.asm.org/ on May 28, 2017 by guest ABSTRACT 22 We tested the biofilm formation potential of 30 heat resistant and six heat sensitive E 23 coli dairy isolates The production of curli and cellulose, static biofilm formation on 24 polystyrene (PS) and stainless steel, formation of biofilm under dynamic conditions 25 (Bioflux), and initial adhesion rates (IAR) were evaluated Biofilm formation varied 26 greatly between strains, media and assay Our results highlight the importance of the 27 experimental setup to determine biofilm formation under conditions of interest, as 28 correlation between different assays was often not given The heat resistant, 29 multidrug resistant (MDR) strain FAM21845 showed the strongest biofilm formation 30 on PS, highest IAR, and was the only one forming significant biofilm on stainless 31 steel under dairy industry relevant conditions and was therefore fully sequenced Its 32 chromosome is 4.9 Mb in size and it harbors a total of five plasmids (147.2, 54.2, 5.8, 33 2.5, and 1.9 kb) The strain encodes a broad range of antimicrobial resistance and 34 biofilm relevant genes, some on its two large conjugative plasmids, as demonstrated 35 in plate mating assays 36 Importance In biofilms, cells are embedded in an extracellular matrix that protects 37 them from stresses like UV radiation, osmotic shock, desiccation, antibiotics and 38 predation Biofilm formation is a major bacterial persistence factor of great concern to 39 the clinic and food industry Many tested strains formed strong biofilm and especially 40 strains like the heat resistant, MDR FAM21845 may pose a serious issue for food 41 production Strong biofilm formation, combined with diverse resistances (some on 42 conjugative plasmids), may allow for increased persistence, co-selection and 43 possible transfer of these resistance factors Horizontal gene transfer could 44 conceivably occur in the food production setting or the gastrointestinal tract after 45 consumption Downloaded from http://aem.asm.org/ on May 28, 2017 by guest 21 INTRODUCTION 47 As opposed to planktonic cultures normally used in laboratory settings, the 48 predominant bacterial mode of growth in nature is as surface adherent communities 49 called biofilms Bacteria growing in biofilms are embedded in a matrix mainly 50 consisting of extracellular polymeric substances The exact composition of the matrix 51 depends on the bacteria comprising the biofilm, but the major constituents are 52 proteins, nucleic acids, polysaccharides, lipids, and water (1) The biofilm lifestyle 53 protects the bacterial community from environmental stresses like UV radiation, 54 osmotic shock, desiccation, antibiotics (up to 1,000-fold increased resistance), 55 predation by invertebrates and the (human) immune system (2, 3) The metabolic 56 activities of cells in a biofilm vary greatly, with a dormant subset, so-called persister 57 cells, being very difficult to eradicate due to their low metabolic activity (4) For these 58 reasons, biofilms are a major concern not only in clinical settings, where they cause 59 wound infections or colonize medical devices (5), but also in the food industry 60 Formation of biofilms on rubber, polyethylene and stainless steel, glass and other 61 food contact surfaces can severely complicate cleaning procedures, which may lead 62 to outbreaks of foodborne illness (6-8) 63 Heat treatment is a widely-used method for inactivation of microbes While 64 autoclaving ensures killing of even bacterial spores, it is often not a feasible option in 65 both clinical and food industry settings, and milder treatment is needed For this 66 reason, even moderate heat resistance, i.e resistance that is much lower compared 67 to that of spores, is a major concern with regard to de-contamination and bacterial 68 persistence, and can have dire consequences Thermization of raw milk at sub- 69 pasteurization temperatures is used to increase safety, while leaving the natural 70 microbiota and enzymes intact (9) There is also a general trend in consumer 71 behavior of preferring minimally processed food and there are foods (i.e meats and Downloaded from http://aem.asm.org/ on May 28, 2017 by guest 46 fresh produce) that simply cannot be heated sufficiently to reliably kill all pathogens 73 without severe degradation of nutrients (10) This leads to a balancing act between 74 food safety, consumer desire, sensory properties and nutritional quality of food In 75 clinical settings, flexible endoscopes are a prime example of a complex tool (with 76 long, narrow channels where bacteria may form biofilm and remain), which usually 77 cannot be treated with high temperatures and other decontamination procedures are 78 required (11) A thermo-chemical treatment (< 60°C) was ineffective at eradicating a 79 K pneumoniae isolate, which harbored a plasmid borne operon encoding a novel clp 80 ATPase, ClpK, mediating increased heat resistance (12) Only one year later, close 81 homologs of much of the operon, including clpK, were found on the chromosome of 82 Cronobacter sakazakii ATCC29544 and an extensive PCR screening (for clpK and 83 another marker gene, orfI) found similar sequences in other Enterobacteriaceae 84 including E coli (13) Comparative genetic analysis found clpK and its surrounding 85 region (flanked by mobile elements, ~14 kb in size), now termed locus of heat 86 resistance (LHR), in approximately 2% of available E coli whole genome (shotgun) 87 sequences including pathogens and food isolates (14) Several heat resistant strains 88 were found in the course of in vitro characterization of E coli raw milk isolates (15) 89 These strains were further analyzed, confirming increased resistance to sub- 90 pasteurization temperatures in milk (16) as well as boosted survival during ripening of 91 semi-hard raw milk cheese (17) In a recent study, 256 E coli raw milk cheese 92 isolates were screened by PCR for clpK (12) and orfI (13) Ninety-three (36.3%) 93 tested positive for both marker genes, while 24 and nine were single positives, 94 respectively We speculated that a thermal selection pressure might have caused this 95 increased abundance compared to the above mentioned estimated 2% amongst all 96 E coli In addition, double positive strains were phenotypically heat resistant in 97 95.7% of cases, confirming great predictive accuracy of these PCRs (18) In contrast, Downloaded from http://aem.asm.org/ on May 28, 2017 by guest 72 C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an 98 a very recent qPCR study found the LHR in only 0.5% of clinical E coli isolates (n = 99 613, (19)), which further supports the speculation of a selective pressure in the dairy 100 environment For the purpose of this study, heat resistance refers to that mediated by 101 clpK and the LHR only A combination of both strong biofilm formation potential and heat resistance 103 can further exacerbate the problem of bacterial persistence Biofilm formation can 104 further protect heat resistant K pneumoniae from heat shock (20) Also, heat 105 resistant, extended-spectrum β-lactamase (ESBL) harboring K pneumoniae have 106 recently caused a nosocomial outbreak (21) Persistent intramammary E coli 107 infections in dairy cows are likely due to biofilm formation of the infecting strains (22) 108 and biofilms can be found on many contact surfaces in the food industry (23) Should 109 such strains be heat resistant as well, increased contamination of raw milk products 110 is to be expected (16, 17) This may then lead to increased incidence of early blowing 111 of cheese (24), or recalls due to exceeding limits on coliform CFU set by hygienic 112 standards We have as of yet not isolated pathogenic, heat resistant E coli from dairy 113 products However, our previous studies demonstrated the possibility of transfer of 114 Shiga-toxin encoding phage and ESBL plasmids to heat resistant E coli (18) and of 115 both LHR of heat resistant dairy isolate FAM21805 to other E coli including 116 pathogenic strains (25) As biofilm formation typically increases rates of horizontal 117 gene transfer (26), it is also of concern in this context 118 We evaluated the biofilm formation potential of 30 heat resistant and six heat 119 sensitive E coli isolated from raw milk and raw milk cheese as well as E coli K-12 120 MG1655 as a reference A large number of phenotypic/functional assays were used 121 to determine biofilm formation under both static (crystal violet (CV) assays in 96well 122 PS plates and on stainless steel coupons (SSC)) and dynamic conditions (initial Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Downloaded from http://aem.asm.org/ on May 28, 2017 by guest 102 C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an adhesion rates and Bioflux flow cells (FC)) Macrocolony assays were employed to 124 qualitatively assess production of curli and cellulose, two major components of the E 125 coli biofilm matrix The strongest overall biofilm former under static conditions and the 126 only isolate able to form biofilm on SSC was FAM21845 We therefore fully 127 sequenced and assembled the genome of this isolate It was analyzed with respect 128 to its LHR, biofilm formation genes and was also found to carry multiple antimicrobial 129 resistance (AMR) and heavy metal resistance genes We detected the presence of 130 five plasmids, including one harboring several AMR genes, a heavy metal resistance 131 operon and a disinfectant resistance gene, and one containing a TEM-1 β-lactamase 132 and the mrkABCDF operon (encoding type III fimbriae), known to increase biofilm 133 formation Conjugation of the mrk containing plasmid into K-12 MG1655 resulted in 134 higher biofilm production, underlining the potential threat of strains like FAM21845 in 135 the food industry The combination of these many resistance and persistence factors 136 is problematic, as co-selection can lead to retaining all of them, even if selection 137 pressure were only applied to one Strains like FAM21845 give rise to another 138 concern: the possible spread of resistance and persistence factors in the food 139 industry Also, transfer of antimicrobial resistance genes in the gastrointestinal tract 140 of humans and in animal models have been observed (reviewed in (27, 28)) and a 141 spread via this route cannot be excluded 142 Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Downloaded from http://aem.asm.org/ on May 28, 2017 by guest 123 C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an RESULTS 144 All strains have phenotypically active rpoS 145 Overall, we tested 37 E coli strains for their potential biofilm formation under a 146 variety of conditions in this study These included 30 heat resistant and six heat 147 sensitive strains isolated from raw milk and raw milk cheese, as well as E coli K-12 148 MG1655 as a further heat sensitive strain and reference Strains were considered 149 heat resistant if their reduction in CFU after 30 incubation at 55°C was less than 150 log Notably, two heat sensitive isolates are ESBL producers (Table 1) As biofilm 151 formation of E coli is influenced by the stationary phase sigma factor rpoS (29), we 152 first tested the activity of catalase as an indirect method to confirm rpoS mediated 153 transcription (30, 31) All 37 strains tested positive for catalase activity and should in 154 turn encode functional, active rpoS (30, 31) 155 Curli and cellulose production 156 Amyloid curli fibers and cellulose are two major components of the E coli biofilm 157 extracellular matrix (32) Macrocolonies are a form of biofilms, and assume structured 158 morphologies dependent on the production of these two matrix constituents 159 Production of both curli fibers (curli) and cellulose results in highly structured colonies 160 with network-like appearance, while high amounts of curli without cellulose result in 161 colonies with concentric wrinkled rings (33) Congo red (staining both curli and 162 cellulose (34)) and calcofluor (staining cellulose (35)) further aid in the qualitative 163 evaluation of production of these two matrix components In most but not all E coli 164 strains, the regulator CsgD, mediating expression of both curli and cellulose is 165 expressed below 30°C (35) It was found that for K-12 strains, CsgD production is 166 higher, resulting in more curli and cellulose production, when they are grown on salt- 167 free LB plates (36) In general, the combination of temperature below 30°C and salt- 168 free media is expected to result in the strongest production of both curli and cellulose Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Downloaded from http://aem.asm.org/ on May 28, 2017 by guest 143 C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an 169 (37) We grew macrocolonies on regular and salt-free LB agar plates (in addition to 170 ABTCAA and RPSMdil agar) at both 28 and 37°C, to assess the impact of salt and 171 temperature on curli and cellulose production in the present strains collective Images of macrocolonies for all 37 strains at 28 and 37°C in all media tested in 173 this study are given in Fig S1 We found three main behaviors regarding curli and 174 cellulose expression at 28 vs 37°C and with salt vs salt-free LB: 1) double positive, 175 no changes; 2) double negative, no changes and 3) double positive, with strong 176 changes due to temperature and salt content (Fig 1) LBnoS at 28°C resulted in the 177 most curli / cellulose double positive macrocolonies (nine) This number was reduced 178 to five at 37°C For LB it was eight at 28°C and two at 37°C and in ABTCAA seven at 179 28°C and one at 37°C Neither curli nor cellulose was produced on RPSMdil agar at 180 either temperature by any strain tested (Table S1) The switch from 28 to 37°C 181 reduced production of curli and/or cellulose in seven isolates on LBnoS, 15 in LB and 182 12 in ABTCAA FAM21843 is the only strain which produced both matrix constituents 183 in all media at both temperatures The production of curli and cellulose showed 184 significant correlations for all media and temperatures tested with the lowest 185 correlation coefficient of 0.478 in ABTCAA at 37°C (Table S2) 186 Biofilm formation on polystyrene surface 187 We tested all 37 E coli strains for biofilm formation on PS surface in LB, LBnoS, 188 RPSMdil and ABTCAA media at 12, 28, and 37°C using CV assays A clear 189 dependency of the extent of biofilm formation on both medium and temperature was 190 observed (Table 2) For each medium except RPSMdil, 28°C led to the greatest 191 overall score of biofilm formation RPSMdil also showed by far the lowest overall 192 score at 12°C In contrast to the other three media, there was no strain which did not 193 form biofilm in RPSMdil It is also interesting to note that 37°C, while being the 194 temperature with the lowest overall biofilm formation for the rich media LB and Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Downloaded from http://aem.asm.org/ on May 28, 2017 by guest 172 C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an 195 LBnoS, was the best and second best temperature for RPSMdil and ABTCAA 196 respectively The best overall medium for biofilm formation at each individual 197 temperature tested was ABTCAA As with media and temperatures, there were large variations in biofilm 199 formation between strains Overall scores per strain ranged from four to 42, with K-12 200 MG1655 in the mid-range (score 24) Only seven strains had a score ≥ under all 201 conditions tested (FAM21845, FAM22954, FAM22961, FAM22963, FAM23016, 202 FAM23030, and FAM23109, Table 2) Variability of biofilm formation (measured by 203 standard deviation of all category values; ‘-‘ = 0), was twice as high for FAM19195 204 (1.60) than for FAM21845 (0.65), demonstrating clear differences of the extent of 205 biofilm formation regulation under the conditions tested Some strains, like 206 FAM22996, were consistent in their lack of biofilm formation over almost all media 207 and temperatures tested (overall score = 4, SD = 0.85) 208 Strains with overall low biofilm formation scores tended to form very little to no 209 biofilm in LB and LBnoS Some weak biofilm formers (FAM22996, FAM21808, 210 FAM22321) also formed little or no biofilm in ABTCAA (Table 2) FAM21845 had the 211 highest overall score of 42 (2nd: FAM22954 with 34), being the most consistent 212 biofilm former (Table 2) although it did not always have the highest absolute OD / 213 ODc ratio At 28°C, we found positive correlations between cellulose production and 214 CV score in all media, and in ABTCAA (28°C) for curli production and CV score as 215 well (Table S2) 216 Initial adhesion rates on polyvinyl chloride 217 The first step in biofilm formation is the initial adhesion of cells to a solid surface, 218 which is then followed by maturation if conditions allow (38) To assess this critical 219 step, we performed IAR measurements for six strains (Fig 2) IAR varied by more Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Downloaded from http://aem.asm.org/ on May 28, 2017 by guest 198 C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an than a factor of ten, from 1.2 × 103 for FAM22321 to 1.7 × 104 cells / (min × cm2) for 221 FAM21845 One-Way ANOVA found significant differences between three groups of 222 strains, with FAM21845 exhibiting the highest IAR, followed by FAM21805 (Fig 2) 223 Biofilm formation under dynamic conditions 224 Biofilms may not only form under static conditions, as tested in our CV assays on PS 225 Dynamism in the surrounding media changes conditions for biofilm formation by 226 introducing shear forces and delivering fresh media to adherent cells (no nutrient 227 depletion) (39) To assess biofilm formation under dynamic conditions, we used the 228 Fluxion Bioflux system As ABTCAA was found to be the overall best media for 229 biofilm formation under static conditions, this media was chosen for dynamic biofilm 230 formation assays For technical reasons, experiments were run for 24 instead of 48 h, 231 and 37°C was used to allow for sufficient growth, even though overall scores were 232 slightly higher at 28°C under static conditions (overall score 114 vs 97) Due to 233 variation between replicates, strains had to be categorized into two sets with overall 234 either good or poor reproducibility Good reproducibility was assigned to strains that 235 consistently either did or did not form biofilm on the sides of the channel and/or within 236 the FC channel between replicates Poor reproducibility was defined as biofilm 237 formation in some, but not all replicates (Table S1) All strains were able to produce 238 biofilm on the walls of the channel (poor reproducibility in FAM21808, FAM22954, 239 and FAM22996) A bacterial lawn, defined as light gray coverage clearly darker than 240 the negative control, but much less dense than true biofilm covering the bottom of the 241 channel, was formed by all strains (poor reproducibility in FAM21808, FAM23078, 242 FAM23106, and FAM22996, Fig 3A) A total of 11 strains showed good 243 reproducibility and consistently produced biofilm within the FC channel Average area 244 coverage percentage graphs of these are given (Fig 3C&D) Importantly, area 245 coverage percentage varied greatly between replicates for most strains, even if they 10 Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Downloaded from http://aem.asm.org/ on May 28, 2017 by guest 220 C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an 966 82 Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, 967 Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, 968 Cardona A 2012 Fiji: an open-source platform for biological-image analysis Nat Methods 969 9:676-682 970 83 Richter AM, Povolotsky TL, Wieler LH, Hengge R 2014 Cyclic-di-GMP signalling and biofilmrelated properties of the Shiga toxin-producing 2011 German outbreak Escherichia coli 972 O104:H4 EMBO Mol Med doi:10.15252/emmm.201404309 973 84 Remus-Emsermann MNP, Schmid M, Gekenidis M-T, Pelludat C, Frey JE, Ahrens CH, 974 Drissner D 2016 Complete genome sequence of Pseudomonas citronellolis P3B5, a 975 candidate for microbial phyllo-remediation of hydrocarbon-contaminated sites Stand 976 Genomic Sci 11:75 977 85 978 979 circularization of genome assemblies using long sequencing reads Genome Biol 16:294 86 980 981 87 Antipov D, Hartwick N, Shen M, Raiko M, Lapidus A, Pevzner PA 2016 plasmidSPAdes: assembling plasmids from whole genome sequencing data Bioinformatics 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77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Downloaded from http://aem.asm.org/ on May 28, 2017 by guest 971 C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an 992 91 Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, Lund O, Aarestrup FM, 993 Larsen MV 2012 Identification of acquired antimicrobial resistance genes J Antimicrob 994 Chemother 67:2640-2644 995 92 Carattoli A, Zankari E, Garcia-Fernandez A, Voldby Larsen M, Lund O, Villa L, Moller 996 Aarestrup F, Hasman H 2014 In silico detection and typing of plasmids using PlasmidFinder 997 and plasmid multilocus sequence typing Antimicrob Agents Chemother 58:3895-3903 93 999 Ponten T, Ussery DW, Aarestrup FM, Lund O 2012 Multilocus sequence typing of total- 1000 1001 Larsen MV, Cosentino S, Rasmussen S, Friis C, Hasman H, Marvig RL, Jelsbak L, Sicheritz- genome-sequenced bacteria J Clin Microbiol 50:1355-1361 94 Gupta SK, Padmanabhan BR, Diene SM, Lopez-Rojas R, Kempf M, Landraud L, Rolain JM 1002 2014 ARG-ANNOT, a new bioinformatic tool to discover antibiotic resistance genes in 1003 bacterial genomes Antimicrob Agents Chemother 58:212-220 1004 95 Moller AK, Leatham MP, Conway T, Nuijten PJ, de Haan LA, Krogfelt KA, Cohen PS 2003 An 1005 Escherichia coli MG1655 lipopolysaccharide deep-rough core mutant grows and survives in 1006 mouse cecal mucus but fails to colonize the mouse large intestine Infect Immun 71:2142- 1007 2152 1008 96 1009 1010 Kuhnert P, Nicolet J, Frey J 1995 Rapid and accurate identification of Escherichia coli K-12 strains Appl Environ Microbiol 61:4135-4139 97 CLSI 2009 Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow 1011 Aerobically; Approved Standard, Eighth Edition CLSI document M07-A8 Wayne, PA: Clinical 1012 and Laboratory Standards Institute 1013 1014 98 Gonzalez-Leiza SM, de Pedro MA, Ayala JA 2011 AmpH, a bifunctional DD-endopeptidase and DD-carboxypeptidase of Escherichia coli J Bacteriol 193:6887-6894 41 Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Downloaded from http://aem.asm.org/ on May 28, 2017 by guest 998 C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an TABLE E coli strains used in this study b Phylogenetic a group FAM19195 O8:H21 B1 heat resistant FAM21805 O68:H14 A heat resistant FAM21807 FAM21808 FAM21843 O68:H14 O11:H11 O178:H12 A A A heat resistant heat resistant heat resistant FAM21845 FAM22636 FAM22639 FAM22791 FAM22808 FAM22891 O68:H14 A A A A A B1 heat resistant heat resistant heat resistant heat resistant heat resistant heat resistant GEN, KAN, STR, CHL, TET, TMP, SXT, AMP str amp, cef ST1434 FAM22936 FAM22940 A B1 heat resistant heat resistant FAM22947 FAM22954 FAM22961 FAM22962 FAM22963 FAM23012 FAM23014 FAM23016 FAM23030 FAM23031 FAM23078 FAM23092 FAM23093 FAM23101 FAM23106 A A A A A A A A A A A A A A A heat resistant heat resistant heat resistant heat resistant heat resistant heat resistant heat resistant heat resistant heat resistant heat resistant heat resistant heat resistant heat resistant heat resistant heat resistant FAM23109 A heat resistant Antibiotic resistance profiles d Serovar MLST Misc c Strain Dairy origin Reference raw milk cheese (15, 16) raw milk cheese (15, 16) str str STR, TMP raw milk cheese raw milk cheese raw milk cheese (15) (15) (15, 16) GEN, KAN, STR, TET, TMP, SXT, AMP str str raw milk cheese raw milk cheese raw milk cheese raw milk cheese raw milk cheese raw milk cheese (15) (18) (18) (18) (18) (18) str raw milk cheese raw milk cheese (18) (18) str str TET TET GEN, KAN, TET, TMP, SXT, AMP str raw milk cheese raw milk cheese raw milk cheese raw milk cheese raw milk cheese raw milk cheese raw milk cheese raw milk cheese raw milk cheese raw milk cheese raw milk cheese raw milk cheese raw milk cheese raw milk cheese raw milk cheese (18) (18) (18) (18) (18) (18) (18) (18) (18) (18) (18) (18) (18) (18) (18) STR, TET, AMP raw milk cheese (18) TET str str 42 Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Downloaded from http://aem.asm.org/ on May 28, 2017 by guest 1015 C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an FAM23113 FAM21846 FAM22942 FAM22956 FAM22996 FAM22321 FAM22871 Serovar O16:H21 Phylogenetic a group A A B1 B1 A A E b MLST ST4483 ST69, STC69 K-12 MG1655 K-12 MG1655 r r NAL , RIF Misc c Antibiotic resistance profiles d heat resistant heat sensitive heat sensitive heat sensitive heat sensitive heat sensitive, ESBL phenotype; TEM-1, CTX-M-14 KAN, STR, CHL, TET, AMP, cef, amc heat sensitive, ESBL phenotype; TEM-1, CTX-M-15 heat sensitive heat sensitive; spontaneous resistant mutant GEN, KAN, STR, TET, AMP, CEF, CXM, CTX, ATM STR, CHL, TET, AMP, cef str TET, AMP GEN, KAN, STR, CHL, TET, NAL, CIP, TMP, SXT, AMP, CEF, CXM, CTX, atm Dairy origin Reference raw milk cheese raw milk cheese raw milk cheese raw milk cheese raw milk cheese raw milk (18) Present Study Present Study Present Study Present Study (18) raw milk NAL, RIF (18) DSM 18039 (95) 1016 a Determined by quadruplex and group C and E specific PCRs (76) 1017 b allele multi locus sequence typing scheme (MLST Database at UoW, (77)) 1018 c all strains tested positive for catalase activity; heat resistant: clpK and orfI positive by PCR and phenotypically heat resistant (< 1log reduction in 1019 CFU after 30 incubation at 55°C (18)) 1020 d 1021 phenotypes respectively Antimicrobials tested: gentamicin (GEN), kanamycin (KAN), streptomycin (STR), chloramphenicol (CHL), 1022 tetracycline (TET), nalidixic acid (NAL), ciprofloxacin (CIP), trimethoprim (TMP), sulfamethoxazol/trimethoprim (19/1, SXT), ampicillin 1023 (AMP), cefoxitin (FOX), cephalothin (CEF), cefuroxime (CXM), cefotaxime (CTX), cefepime (FEP), aztreonam (ATM), 1024 amoxicillin/clavulanic acid (20/10, AMC) and ertapenem (ETP) Antimicrobial resistances determined according to CLSI guidelines (78); capital and minor letters indicate resistant and intermediate 43 Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Downloaded from http://aem.asm.org/ on May 28, 2017 by guest Strain C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an TABLE Static biofilm formation on PS and SSC after 48 h incubation and biofilm formation in channel of FC after 24 h Strain FAM19195 FAM21805 FAM21807 FAM21808 FAM21843 FAM21845 FAM22636 FAM22639 FAM22791 FAM22808 FAM22891 FAM22936 FAM22940 FAM22947 FAM22954 FAM22961 FAM22962 FAM22963 FAM23012 FAM23014 FAM23016 FAM23030 FAM23031 FAM23078 FAM23092 FAM23093 FAM23101 FAM23106 FAM23109 FAM23113 FAM21846 FAM22942 FAM22956 FAM22996 FAM22321 FAM22871 K-12 MG1655 96well, polystyrene (PS) RPSMdil [°C] 12 28 37 3 3 3 3 2 3 3 3 2 3 4 3 3 3 2 3 2 3 2 2 2 2 2 2 3 3 2 Misc HR HR HR HR HR HR HR HR HR HR HR HR HR HR HR HR HR HR HR HR HR HR HR HR HR HR HR HR HR HR HS HS HS HS HS, ESBL HS, ESBL HS 12 1 3 2 3 3 1 1 LB [°C] 28 4 3 2 2 1 3 3 3 1 3 3 4 4 37 2 2 1 2 1 2 1 2 2 4 1 12 3 2 3 3 3 4 1 1 LBnoS [°C] 28 3 4 2 1 3 2 4 1 3 2 4 37 1 2 1 2 2 2 1 2 2 1 Overall score 61 92 55 60 85 51 90 95 ABTCAA [°C] 12 28 37 3 1 4 4 3 2 3 3 2 3 4 4 4 3 3 4 4 1 3 3 4 3 3 3 3 4 3 1 2 75 114 Overall score 20 23 19 30 42 19 26 23 24 27 27 13 24 34 28 11 32 31 30 33 30 16 15 30 28 33 14 26 30 15 31 31 10 22 24 97 SSC RPSM [°C] 12 1 positive FC ABTCAA [°C] 37 + + + + + + + + + + + 11 positives 1026 Numbers in columns indicate categories defined by OD to ODc ratios (79) Cut-off value: ODc = average ODnegative 1027 SD(ODnegative control) The categories are: OD ≤ ODc: -; ODc < OD ≤ × ODc: 1; × ODc < OD ≤ × ODc: 2; × ODc< OD ≤ × ODc: control + × 44 Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn Downloaded from http://aem.asm.org/ on May 28, 2017 by guest 1025 C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an 3; × ODc