259607018aa783a44d956f88e08bdd08 biomolecules_a2021v11n3p475.pdf 7d3e9bdbafd7a30aef1148884754f5a241e385e0 biomolecules_a2021v11n3p475.pdf 39be527190f7a2ee7571040445f2af275c0e9f602b7d3c4cdb6f1f1b9a7ac9c8 biomolecules_a2021v11n3p475.pdf Title: In Vitro Preformed Biofilms of Bacillus safensis Inhibit the Adhesion and Subsequent Development of Listeria monocytogenes on Stainless-Steel Surfaces Subject: Listeria monocytogenes continues to be one of the most important public health challenges for the meat sector. Many attempts have been made to establish the most efficient cleaning and disinfection protocols, but there is still the need for the sector to develop plans with different lines of action. In this regard, an interesting strategy could be based on the control of this type of foodborne pathogen through the resident microbiota naturally established on the surfaces. A potential inhibitor, Bacillus safensis, was found in a previous study that screened the interaction between the resident microbiota and L. monocytogenes in an Iberian pig processing plant. The aim of the present study was to evaluate the effect of preformed biofilms of Bacillus safensis on the adhesion and implantation of 22 strains of L. monocytogenes. Mature preformed B. safensis biofilms can inhibit adhesion and the biofilm formation of multiple L. monocytogenes strains, eliminating the pathogen by a currently unidentified mechanism. Due to the non-enterotoxigenic properties of B. safensis, its presence on certain meat industry surfaces should be favored and it could represent a new way to fight against the persistence of L. monocytogenes in accordance with other bacterial inhibitors and hygiene operations. Keywords: biofilms; Listeria monocytogenes; pathogen inhibition; microbiota; surfaces; Bacillus safensis Author: Anne-Sophie Hascoët, Carolina Ripolles-Avila, Brayan R. H. Cervantes-Huamán and José Juan Rodríguez-Jerez Creator: LaTeX with hyperref package Producer: pdfTeX-1.40.18 CreationDate: Wed Mar 24 03:07:08 2021 CET ModDate: Wed Mar 24 04:31:28 2021 CET Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 16 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3541311 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- PMCKNV+URWPalladioL-Ital Type 1 Custom yes yes no 10 0 RPFJRQ+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 MBVIXA+URWPalladioL-BoldItal Type 1 Custom yes yes no 19 0 SIAIVO+URWPalladioL-Roma Type 1 Custom yes yes no 23 0 IOJCNK+VnURWPalladioL-Bold Type 1 Custom yes yes no 27 0 MWHIBA+VnURWPalladioL Type 1 Custom yes yes no 32 0 IVVSWS+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 42 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 72 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 76 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 80 0 CAMXTK+EURM10 Type 1 Builtin yes yes no 92 0 IXLVLQ+CMSY10 Type 1 Builtin yes yes no 99 0 RDUGED+VnURWPalladioL-Italic Type 1 Custom yes yes no 155 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-10-02 03:52:30 CEST RepresentationInformation: biomolecules_a2021v11n3p475.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2023-03-31 11:50:49 CEST Size: 3541311 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 373 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Evaluation of the Ability of B. safensis to Form Biofilms Destination: subsection.2.1 Children: Item: Title: Study Surface Destination: subsubsection.2.1.1 Item: Title: Inoculum Preparation Destination: subsubsection.2.1.2 Item: Title: Formation of Monospecies Biofilms In Vitro Destination: subsubsection.2.1.3 Item: Title: Evaluation of Biofilm Formation through DEM Destination: subsubsection.2.1.4 Item: Title: Evaluation of the Effect of Preformed B. safensis Biofilms on 22 Strains of L. monocytogenes Destination: subsection.2.2 Children: Item: Title: L. monocytogenes Strains Destination: subsubsection.2.2.1 Item: Title: Inoculums Preparation Destination: subsubsection.2.2.2 Item: Title: In Vitro Formation of Monospecies Biofilms and Subsequent Implantation of L. monocytogenes on the Structure Destination: subsubsection.2.2.3 Item: Title: Quantification of Biofilms by ALOA, TEMPO, and qPCR Destination: subsubsection.2.2.4 Item: Title: Evaluation of the Effect of Preformed Biofilms of Pseudomonas spp. and Candida zeylanoides on L. monocytogenes Destination: subsection.2.3 Item: Title: Characterization of the Effect of Preformed B. safensis Biofilms on L. monocytogenes Destination: subsection.2.4 Item: Title: Statistical Analysis Destination: subsection.2.5 Item: Title: Results and Discussion Destination: section.3 Children: Item: Title: Evaluation of the Ability of B. safensis Biofilm Formation Destination: subsection.3.1 Item: Title: Evaluation of the Effect of B. safensis Preformed Biofilms on 22 Strains of L. monocytogenes Destination: subsection.3.2 Item: Title: Evaluation of the Effect of Preformed Biofilms of Pseudomonas spp. and C. zeylanoides on L. monocytogenes Destination: subsection.3.3 Item: Title: Characterization of the Effect of Preformed Biofilms of B. safensis on L. monocytogenes Destination: subsection.3.4 Item: Title: Conclusions Destination: section.4 Item: Title: References Destination: section.4 Info: Title: In Vitro Preformed Biofilms of Bacillus safensis Inhibit the Adhesion and Subsequent Development of Listeria monocytogenes on Stainless-Steel Surfaces Author: Anne-Sophie Hascoët, Carolina Ripolles-Avila, Brayan R. H. Cervantes-Huamán and José Juan Rodríguez-Jerez Subject: Listeria monocytogenes continues to be one of the most important public health challenges for the meat sector. Many attempts have been made to establish the most efficient cleaning and disinfection protocols, but there is still the need for the sector to develop plans with different lines of action. In this regard, an interesting strategy could be based on the control of this type of foodborne pathogen through the resident microbiota naturally established on the surfaces. A potential inhibitor, Bacillus safensis, was found in a previous study that screened the interaction between the resident microbiota and L. monocytogenes in an Iberian pig processing plant. The aim of the present study was to evaluate the effect of preformed biofilms of Bacillus safensis on the adhesion and implantation of 22 strains of L. monocytogenes. Mature preformed B. safensis biofilms can inhibit adhesion and the biofilm formation of multiple L. monocytogenes strains, eliminating the pathogen by a currently unidentified mechanism. Due to the non-enterotoxigenic properties of B. safensis, its presence on certain meat industry surfaces should be favored and it could represent a new way to fight against the persistence of L. monocytogenes in accordance with other bacterial inhibitors and hygiene operations. Keywords: biofilms; Listeria monocytogenes; pathogen inhibition; microbiota; surfaces; Bacillus safensis Creator: LaTeX with hyperref package Producer: pdfTeX-1.40.18 CreationDate: Wed Mar 24 03:07:08 CET 2021 ModDate: Wed Mar 24 04:31:28 CET 2021 ID: 0xcc295113cf954c248eb4f8c478bc9009, 0xcc295113cf954c248eb4f8c478bc9009 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: MWHIBA+VnURWPalladioL FontSubset: true FirstChar: 161 LastChar: 226 FontDescriptor: FontName: MWHIBA+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: MBVIXA+URWPalladioL-BoldItal FontSubset: true FirstChar: 66 LastChar: 121 FontDescriptor: FontName: MBVIXA+URWPalladioL-BoldItal Flags: Symbolic FontBBox: -170, -300, 1073, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: IXLVLQ+CMSY10 FontSubset: true FirstChar: 2 LastChar: 14 FontDescriptor: FontName: IXLVLQ+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: SIAIVO+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 248 FontDescriptor: FontName: SIAIVO+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: PMCKNV+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 122 FontDescriptor: FontName: PMCKNV+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: IOJCNK+VnURWPalladioL-Bold FontSubset: true FirstChar: 161 LastChar: 189 FontDescriptor: FontName: IOJCNK+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: RDUGED+VnURWPalladioL-Italic FontSubset: true FirstChar: 161 LastChar: 226 FontDescriptor: FontName: RDUGED+VnURWPalladioL-Italic Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: CAMXTK+EURM10 FontSubset: true FirstChar: 12 LastChar: 22 FontDescriptor: FontName: CAMXTK+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: RPFJRQ+URWPalladioL-Bold FontSubset: true FirstChar: 2 LastChar: 235 FontDescriptor: FontName: RPFJRQ+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 35, 759, 173, 795 Annotation: Subtype: Link Rect: 515, 759, 560, 795 Annotation: Subtype: Link Rect: 248, 640, 259, 654 Annotation: Subtype: Link Rect: 180, 628, 191, 641 Annotation: Subtype: Link Rect: 149, 347, 154, 355 Annotation: Subtype: Link Rect: 35, 334, 159, 343 Annotation: Subtype: Link Rect: 35, 162, 97, 184 Annotation: Subtype: Link Rect: 153, 89, 154, 98 Annotation: Subtype: Link Rect: 35, 77, 147, 86 Annotation: Subtype: Link Rect: 285, 273, 292, 282 ActionDest: 149 Annotation: Subtype: Link Rect: 292, 273, 299, 282 ActionDest: 149 Annotation: Subtype: Link Rect: 465, 273, 472, 282 ActionDest: 149 Annotation: Subtype: Link Rect: 193, 248, 200, 257 ActionDest: 149 Annotation: Subtype: Link Rect: 402, 223, 409, 232 ActionDest: 149 Annotation: Subtype: Link Rect: 409, 223, 416, 232 ActionDest: 149 Annotation: Subtype: Link Rect: 481, 210, 488, 219 ActionDest: 149 Annotation: Subtype: Link Rect: 541, 185, 548, 194 ActionDest: 149 Annotation: Subtype: Link Rect: 549, 185, 556, 194 ActionDest: 149 Annotation: Subtype: Link Rect: 400, 110, 407, 119 ActionDest: 149 Annotation: Subtype: Link Rect: 410, 110, 417, 119 ActionDest: 149 Annotation: Subtype: Link Rect: 333, 97, 340, 106 ActionDest: 149 Annotation: Subtype: Link Rect: 430, 97, 437, 106 ActionDest: 149 Annotation: Subtype: Link Rect: 544, 97, 556, 106 ActionDest: 149 Annotation: Subtype: Link Rect: 126, 17, 269, 28 Annotation: Subtype: Link Rect: 388, 17, 558, 28 Annotation: Subtype: Link Rect: 35, 446, 87, 465 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 218, 745, 225, 754 ActionDest: 149 Annotation: Subtype: Link Rect: 407, 682, 419, 691 ActionDest: 149 Annotation: Subtype: Link Rect: 420, 682, 432, 691 ActionDest: 149 Annotation: Subtype: Link Rect: 247, 607, 259, 616 ActionDest: 149 Annotation: Subtype: Link Rect: 260, 606, 272, 616 ActionDest: 149 Annotation: Subtype: Link Rect: 400, 569, 407, 578 ActionDest: 149 Annotation: Subtype: Link Rect: 316, 531, 328, 540 ActionDest: 149 Annotation: Subtype: Link Rect: 430, 519, 442, 528 ActionDest: 149 Annotation: Subtype: Link Rect: 190, 481, 202, 490 ActionDest: 149 Annotation: Subtype: Link Rect: 247, 443, 259, 452 ActionDest: 149 Annotation: Subtype: Link Rect: 262, 443, 274, 452 ActionDest: 152 Annotation: Subtype: Link Rect: 544, 343, 556, 352 ActionDest: 152 Annotation: Subtype: Link Rect: 200, 318, 212, 327 ActionDest: 152 Annotation: Subtype: Link Rect: 247, 305, 259, 314 ActionDest: 152 Annotation: Subtype: Link Rect: 411, 255, 422, 264 ActionDest: 149 Annotation: Subtype: Link Rect: 544, 230, 556, 239 ActionDest: 149 Annotation: Subtype: Link Rect: 363, 205, 375, 214 ActionDest: 152 Annotation: Subtype: Link Rect: 375, 205, 387, 214 ActionDest: 152 Annotation: Subtype: Link Rect: 519, 180, 531, 189 ActionDest: 152 Annotation: Subtype: Link Rect: 427, 130, 439, 138 ActionDest: 152 Annotation: Subtype: Link Rect: 225, 104, 237, 113 ActionDest: 152 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 418, 732, 430, 741 ActionDest: 152 Annotation: Subtype: Link Rect: 211, 479, 223, 488 ActionDest: 152 Annotation: Subtype: Link Rect: 258, 416, 270, 425 ActionDest: 149 Annotation: Subtype: Link Rect: 304, 177, 316, 186 ActionDest: 152 Annotation: Subtype: Link Rect: 419, 164, 431, 173 ActionDest: 152 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 446, 732, 458, 741 ActionDest: 152 Annotation: Subtype: Link Rect: 438, 516, 445, 528 ActionDest: 103 Annotation: Subtype: Link Rect: 242, 217, 249, 229 ActionDest: 109 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 504, 709, 515, 717 ActionDest: 152 Annotation: Subtype: Link Rect: 504, 687, 515, 695 ActionDest: 152 Annotation: Subtype: Link Rect: 504, 676, 515, 684 ActionDest: 152 Annotation: Subtype: Link Rect: 504, 665, 515, 673 ActionDest: 152 Annotation: Subtype: Link Rect: 504, 654, 515, 662 ActionDest: 152 Annotation: Subtype: Link Rect: 504, 643, 515, 651 ActionDest: 152 Annotation: Subtype: Link Rect: 504, 632, 515, 640 ActionDest: 152 Annotation: Subtype: Link Rect: 504, 621, 515, 629 ActionDest: 152 Annotation: Subtype: Link Rect: 504, 610, 515, 618 ActionDest: 152 Annotation: Subtype: Link Rect: 504, 599, 515, 607 ActionDest: 152 Annotation: Subtype: Link Rect: 504, 587, 515, 596 ActionDest: 152 Annotation: Subtype: Link Rect: 504, 543, 515, 551 ActionDest: 152 Annotation: Subtype: Link Rect: 504, 477, 515, 485 ActionDest: 149 Annotation: Subtype: Link Rect: 289, 421, 310, 433 ActionDest: 96 Annotation: Subtype: Link Rect: 515, 399, 537, 408 ActionDest: 109 Annotation: Subtype: Link Rect: 301, 324, 313, 333 ActionDest: 152 Annotation: Subtype: Link Rect: 322, 162, 334, 171 ActionDest: 152 Annotation: Subtype: Link Rect: 277, 99, 289, 109 ActionDest: 152 Annotation: Subtype: Link Rect: 289, 99, 301, 109 ActionDest: 152 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 220, 606, 232, 615 ActionDest: 149 Annotation: Subtype: Link Rect: 232, 606, 244, 615 ActionDest: 152 Annotation: Subtype: Link Rect: 244, 606, 256, 615 ActionDest: 152 Annotation: Subtype: Link Rect: 257, 606, 269, 615 ActionDest: 152 Annotation: Subtype: Link Rect: 504, 305, 516, 314 ActionDest: 152 Annotation: Subtype: Link Rect: 501, 229, 515, 241 ActionDest: 103 Annotation: Subtype: Link Rect: 196, 194, 208, 203 ActionDest: 149 Annotation: Subtype: Link Rect: 427, 181, 439, 190 ActionDest: 149 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 259, 742, 273, 754 ActionDest: 103 Annotation: Subtype: Link Rect: 438, 417, 450, 426 ActionDest: 152 Annotation: Subtype: Link Rect: 553, 414, 560, 426 ActionDest: 103 Annotation: Subtype: Link Rect: 202, 366, 214, 376 ActionDest: 152 Annotation: Subtype: Link Rect: 214, 367, 226, 376 ActionDest: 152 Annotation: Subtype: Link Rect: 238, 329, 250, 338 ActionDest: 152 Annotation: Subtype: Link Rect: 358, 266, 370, 275 ActionDest: 149 Annotation: Subtype: Link Rect: 517, 150, 525, 162 ActionDest: 121 Annotation: Subtype: Link Rect: 428, 112, 435, 124 ActionDest: 121 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 540, 641, 547, 653 ActionDest: 121 Annotation: Subtype: Link Rect: 521, 569, 533, 578 ActionDest: 152 Annotation: Subtype: Link Rect: 299, 556, 311, 565 ActionDest: 152 Annotation: Subtype: Link Rect: 489, 468, 501, 477 ActionDest: 152 Annotation: Subtype: Link Rect: 528, 305, 540, 314 ActionDest: 159 Annotation: Subtype: Link Rect: 365, 268, 372, 277 ActionDest: 149 Annotation: Subtype: Link Rect: 322, 255, 334, 264 ActionDest: 152 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 232, 80, 244, 89 ActionDest: 152 Annotation: Subtype: Link Rect: 244, 80, 256, 89 ActionDest: 159 Annotation: Subtype: Link Rect: 257, 80, 269, 89 ActionDest: 159 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 449, 744, 461, 754 ActionDest: 159 Annotation: Subtype: Link Rect: 186, 732, 198, 741 ActionDest: 152 Annotation: Subtype: Link Rect: 202, 657, 214, 666 ActionDest: 152 Annotation: Subtype: Link Rect: 544, 644, 556, 653 ActionDest: 159 Annotation: Subtype: Link Rect: 500, 370, 512, 379 ActionDest: 152 Annotation: Subtype: Link Rect: 220, 282, 232, 291 ActionDest: 152 Annotation: Subtype: Link Rect: 401, 257, 413, 266 ActionDest: 159 Annotation: Subtype: Link Rect: 359, 194, 371, 203 ActionDest: 149 Annotation: Subtype: Link Rect: 385, 119, 397, 128 ActionDest: 152 Annotation: Subtype: Link Rect: 398, 119, 410, 128 ActionDest: 159 Annotation: Subtype: Link Rect: 196, 78, 203, 90 ActionDest: 137 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 497, 742, 503, 754 ActionDest: 137 Annotation: Subtype: Link Rect: 288, 692, 295, 704 ActionDest: 137 Annotation: Subtype: Link Rect: 258, 654, 265, 666 ActionDest: 137 Annotation: Subtype: Link Rect: 544, 644, 556, 653 ActionDest: 159 Annotation: Subtype: Link Rect: 191, 569, 203, 578 ActionDest: 159 Annotation: Subtype: Link Rect: 348, 443, 360, 452 ActionDest: 159 Annotation: Subtype: Link Rect: 397, 418, 409, 427 ActionDest: 159 Annotation: Subtype: Link Rect: 504, 406, 516, 415 ActionDest: 159 Annotation: Subtype: Link Rect: 356, 318, 368, 327 ActionDest: 159 Annotation: Subtype: Link Rect: 544, 204, 558, 214 ActionDest: 115 Page: Sequence: 12 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 290, 704, 297, 716 ActionDest: 143 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 158, 518, 195, 528 Annotation: Subtype: Link Rect: 202, 518, 238, 528 Annotation: Subtype: Link Rect: 59, 495, 97, 505 Annotation: Subtype: Link Rect: 103, 495, 139, 505 Annotation: Subtype: Link Rect: 465, 471, 503, 482 Annotation: Subtype: Link Rect: 405, 448, 442, 459 Annotation: Subtype: Link Rect: 82, 415, 119, 425 Annotation: Subtype: Link Rect: 136, 380, 173, 390 Annotation: Subtype: Link Rect: 179, 380, 216, 390 Annotation: Subtype: Link Rect: 320, 345, 357, 356 Annotation: Subtype: Link Rect: 363, 345, 399, 356 Annotation: Subtype: Link Rect: 152, 311, 189, 321 Annotation: Subtype: Link Rect: 171, 277, 209, 287 Annotation: Subtype: Link Rect: 218, 253, 256, 264 Annotation: Subtype: Link Rect: 262, 253, 298, 264 Annotation: Subtype: Link Rect: 59, 230, 97, 241 Annotation: Subtype: Link Rect: 378, 195, 416, 206 Annotation: Subtype: Link Rect: 129, 161, 167, 172 Annotation: Subtype: Link Rect: 291, 138, 329, 149 Annotation: Subtype: Link Rect: 335, 138, 371, 149 Annotation: Subtype: Link Rect: 391, 103, 428, 114 Annotation: Subtype: Link Rect: 378, 80, 416, 91 Annotation: Subtype: Link Rect: 422, 80, 458, 91 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 59, 720, 97, 730 Annotation: Subtype: Link Rect: 103, 720, 139, 730 Annotation: Subtype: Link Rect: 204, 696, 241, 707 Annotation: Subtype: Link Rect: 519, 673, 557, 684 Annotation: Subtype: Link Rect: 251, 639, 288, 650 Annotation: Subtype: Link Rect: 536, 627, 560, 638 Annotation: Subtype: Link Rect: 56, 616, 440, 627 Annotation: Subtype: Link Rect: 416, 582, 453, 592 Annotation: Subtype: Link Rect: 131, 559, 169, 569 Annotation: Subtype: Link Rect: 369, 535, 407, 546 Annotation: Subtype: Link Rect: 413, 535, 449, 546 Annotation: Subtype: Link Rect: 289, 512, 327, 523 Annotation: Subtype: Link Rect: 333, 512, 369, 523 Annotation: Subtype: Link Rect: 283, 478, 320, 489 Annotation: Subtype: Link Rect: 520, 397, 557, 408 Annotation: Subtype: Link Rect: 171, 363, 209, 373 Annotation: Subtype: Link Rect: 493, 339, 530, 350 Annotation: Subtype: Link Rect: 140, 306, 178, 316 Annotation: Subtype: Link Rect: 417, 282, 455, 293 Annotation: Subtype: Link Rect: 393, 259, 431, 270 Annotation: Subtype: Link Rect: 434, 213, 471, 224 Annotation: Subtype: Link Rect: 262, 190, 299, 201 Annotation: Subtype: Link Rect: 265, 167, 303, 178 Annotation: Subtype: Link Rect: 109, 145, 146, 155 Annotation: Subtype: Link Rect: 343, 121, 380, 132 Annotation: Subtype: Link Rect: 427, 98, 464, 109 Annotation: Subtype: Link Rect: 400, 75, 438, 86 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 416, 731, 453, 742 Annotation: Subtype: Link Rect: 479, 719, 515, 730 Annotation: Subtype: Link Rect: 521, 719, 557, 730 Annotation: Subtype: Link Rect: 296, 696, 334, 707 Annotation: Subtype: Link Rect: 340, 696, 376, 707 Annotation: Subtype: Link Rect: 219, 674, 256, 684 Annotation: Subtype: Link Rect: 464, 650, 501, 661 Annotation: Subtype: Link Rect: 277, 627, 314, 638 Annotation: Subtype: Link Rect: 321, 627, 357, 638 Annotation: Subtype: Link Rect: 234, 605, 272, 615 Annotation: Subtype: Link Rect: 59, 570, 97, 581 Checksum: 4de4db4f Type: CRC32 Checksum: 259607018aa783a44d956f88e08bdd08 Type: MD5 Checksum: 7d3e9bdbafd7a30aef1148884754f5a241e385e0 Type: SHA-1 Checksum: 39be527190f7a2ee7571040445f2af275c0e9f602b7d3c4cdb6f1f1b9a7ac9c8 Type: SHA-256