@article{pham_linova_smith_brown_elhanafi_fan_lavoie_woodley_carbonell_2024, title={Novel multimodal cation-exchange membrane for the purification of a single-chain variable fragment from Pichia pastoris supernatant}, volume={1718}, ISSN={["1873-3778"]}, DOI={10.1016/j.chroma.2024.464682}, abstractNote={A novel salt-tolerant cation-exchange membrane, prepared with a multimodal ligand, 2-mercaptopyridine-3-carboxylic acid (MMC-MPCA), was examined for its purification properties in a bind-and-elute mode from the high conductivity supernatant of a Pichia pastoris fermentation producing and secreting a single-chain variable fragment (scFv). If successful, this approach would eliminate the need for a buffer exchange prior to product capture by ion-exchange. Two fed-batch fermentations of Pichia pastoris resulted in fermentation supernatants reaching an scFv titer of 395.0 mg/L and 555.7 mg/L, both with a purity of approximately 83%. The MMC-MPCA membrane performance was characterized in terms of pH, residence time (RT), scFv load, and scFv concentration to identify the resulting dynamic binding capacity (DBC), yield, and purity achieved under optimal conditions. The MMC-MPCA membrane exhibited the highest DBC of 39.06 mg/mL at pH 5.5, with a residence time of 1 minute, while reducing the pH below 5.0 resulted in a significant decrease of the DBC to around 2.5 mg/mL. With almost no diffusional limitations, reducing the RT from 2 to 0.2 min did not negatively impact the DBC of the MMC-MPCA membrane, resulting in a significant improvement in productivity of up to 180 mg/mL/min at 0.2 min RT. Membrane fouling was observed when reusing the membranes at 0.2 and 0.5 min RT, likely due to the enhanced adsorption of impurities on the membrane. Changing the amount of scFv loaded onto the membrane column did not show any changes in yield, instead a 10-20% loss of scFv was observed, which suggested that some of the produced scFv were fragmented or had aggregated. When performing the purification under the optimized conditions, the resulting purity of the product improved from 83% to approximately 92-95%.}, journal={JOURNAL OF CHROMATOGRAPHY A}, author={Pham, Dan N. and Linova, Marina Y. and Smith, William K. and Brown, Hunter and Elhanafi, Driss and Fan, Jinxin and Lavoie, Joseph and Woodley, John M. and Carbonell, Ruben G.}, year={2024}, month={Mar} }
 @article{brown_chen_ivanova_leekitcharoenphon_parsons_niedermeyer_gould_strules_mesa-cruz_kelly_et al._2023, title={Draft Genome Sequences of 158 Listeria monocytogenes Strains Isolated from Black Bears (Ursus americanus) in the United States}, volume={12}, ISSN={2576-098X}, url={http://dx.doi.org/10.1128/mra.00248-23}, DOI={10.1128/mra.00248-23}, abstractNote={
            Listeria monocytogenes
            is responsible for severe foodborne disease and major economic losses, but its potential reservoirs in natural ecosystems remain poorly understood. Here, we report the draft genome sequences of 158
            L. monocytogenes
            strains isolated from black bears (
            Ursus americanus
            ) in the southeastern United States between 2014 and 2017.
          }, number={7}, journal={Microbiology Resource Announcements}, publisher={American Society for Microbiology}, author={Brown, Phillip and Chen, Yi and Ivanova, Mirena and Leekitcharoenphon, Pimlapas and Parsons, Cameron and Niedermeyer, Jeffrey and Gould, Nicholas and Strules, Jennifer and Mesa-Cruz, J. Bernardo and Kelly, Marcella J. and et al.}, editor={Rasko, DavidEditor}, year={2023}, month={Jul} }
 @article{brown_kanenaka_chen_ivanova_leekitcharoenphon_elhanafi_kathariou_2023, title={Draft Genome Sequences of Closely Related Listeria monocytogenes Lineage III Strains Isolated from a Food Processing Environment and a Case of Human Listeriosis}, volume={5}, ISSN={["2576-098X"]}, url={https://doi.org/10.1128/mra.00250-23}, DOI={10.1128/mra.00250-23}, abstractNote={
            Listeria monocytogenes
            lineage III is genetically highly diverse, and closely related lineage III strains from food facilities and human listeriosis have not been reported. Here, we report the genome sequences of three closely related lineage III strains from Hawaii, namely, one isolated from a human case and two isolated from a produce storage facility.
          }, journal={MICROBIOLOGY RESOURCE ANNOUNCEMENTS}, author={Brown, Phillip and Kanenaka, Rebecca and Chen, Yi and Ivanova, Mirena and Leekitcharoenphon, Pimlapas and Elhanafi, Driss and Kathariou, Sophia}, editor={Rasko, DavidEditor}, year={2023}, month={May} }
 @article{brown_murray_galsworthy_ivanova_leekitcharoenphon_ward_kucerova_chen_elhanafi_siletzky_et al._2023, title={Draft genome sequences of a historical collection of Listeria monocytogenes from humans and other sources, 1926-1964}, volume={9}, ISSN={["2576-098X"]}, url={https://doi.org/10.1128/MRA.00625-23}, DOI={10.1128/MRA.00625-23}, abstractNote={ABSTRACT}, journal={MICROBIOLOGY RESOURCE ANNOUNCEMENTS}, author={Brown, Phillip and Murray, Robert G. E. and Galsworthy, Sara and Ivanova, Mirena and Leekitcharoenphon, Pimlapas and Ward, Todd and Kucerova, Zuzana and Chen, Yi and Elhanafi, Driss and Siletzky, Robin and et al.}, editor={Dennehy, John J.Editor}, year={2023}, month={Sep} }
 @article{sadat_farag_elhanafi_awad_elmahallawy_alsowayeh_el-khadragy_elshopakey_2023, title={Immunological and Oxidative Biomarkers in Bovine Serum from Healthy, Clinical, and Sub-Clinical Mastitis Caused by Escherichia coli and Staphylococcus aureus Infection}, volume={13}, ISSN={["2076-2615"]}, DOI={10.3390/ani13050892}, abstractNote={The study aimed to investigate the mastitis’ emerging causative agents and their antimicrobial sensitivity, in addition to the hematological, biochemical indicators, oxidative biomarkers, acute phase protein (APP), and inflammatory cytokine changes in dairy farms in Gamasa, Dakahlia Governorate, Egypt. One hundred Holstein Friesian dairy cattle with clinical and subclinical mastitis were investigated and were allocated into three groups based on a thorough clinical examination. Escherichia coli and Staphylococcus aureus were found responsible for the clinical and subclinical mastitis in dairy farms, respectively. Multiple drug resistance (MDR) was detected in 100%, and 94.74% of E. coli and S. aureus isolates, respectively. Significantly low RBCs count, Hb, and PCV values were detected in mastitic cows compared with both subclinical mastitic and control groups; moreover, WBCs, lymphocytes, and neutrophil counts were significantly diminished in mastitic cows compared to the controls. Significantly higher levels of AST, LDH, total protein, and globulin were noticed in both mastitic and subclinical mastitic cows. The haptoglobin, fibrinogen, amyloid A, ceruloplasmin, TNF-α, IL-1β, and IL-6 levels were statistically increased in mastitic cows compared to the controls. Higher MDA levels and reduction of TAC and catalase were identified in all the mastitic cases compared to the controls. Overall, the findings suggested potential public health hazards due to antimicrobial resistance emergence. Meanwhile, the APP and cytokines, along with antioxidant markers can be used as early indicators of mastitis.}, number={5}, journal={ANIMALS}, author={Sadat, Asmaa and Farag, Alshimaa M. M. and Elhanafi, Driss and Awad, Amal and Elmahallawy, Ehab Kotb and Alsowayeh, Noorah and El-khadragy, Manal F. and Elshopakey, Gehad E.}, year={2023}, month={Mar} }
 @article{brown_lee_elhanafi_tham_danielsson-tham_lopez-valladares_chen_ivanova_leekitcharoenphon_kathariou_2023, title={Investigation of a Listeria monocytogenes Chromosomal Immigration Control Region Reveals Diverse Restriction Modification Systems with Complete Sequence Type Conservation}, volume={11}, ISSN={["2076-2607"]}, url={https://doi.org/10.3390/microorganisms11030699}, DOI={10.3390/microorganisms11030699}, abstractNote={Listeria monocytogenes is a Gram-positive pathogen responsible for the severe foodborne disease listeriosis. A chromosomal hotspot between lmo0301 and lmo0305 has been noted to harbor diverse restriction modification (RM) systems. Here, we analyzed 872 L. monocytogenes genomes to better understand the prevalence and types of RM systems in this region, designated the immigration control region (ICR). Type I, II, III and IV RM systems were found in 86.1% of strains inside the ICR and in 22.5% of strains flanking the ICR. ICR content was completely conserved within the same multilocus sequence typing-based sequence type (ST), but the same RM system could be identified in diverse STs. The intra-ST conservation of ICR content suggests that this region may drive the emergence of new STs and promote clone stability. Sau3AI-like, LmoJ2 and LmoJ3 type II RM systems as well as type I EcoKI-like, and type IV AspBHI-like and mcrB-like systems accounted for all RM systems in the ICR. A Sau3AI-like type II RM system with specificity for GATC was harbored in the ICR of many STs, including all strains of the ancient, ubiquitous ST1. The extreme paucity of GATC recognition sites in lytic phages may reflect ancient adaptation of these phages to preempt resistance associated with the widely distributed Sau3AI-like systems. These findings indicate that the ICR has a high propensity for RM systems which are intraclonaly conserved and may impact bacteriophage susceptibility as well as ST emergence and stability.}, number={3}, journal={MICROORGANISMS}, author={Brown, Phillip and Lee, Sangmi and Elhanafi, Driss and Tham, Wilhelm and Danielsson-Tham, Marie-Louise and Lopez-Valladares, Gloria and Chen, Yi and Ivanova, Mirena and Leekitcharoenphon, Pimlapas and Kathariou, Sophia}, year={2023}, month={Mar} }
 @article{sripada_elhanafi_collins_williams_linova_woodley_boi_menegatti_2023, title={Pseudo-affinity capture of <i>K. phaffii</i> host cell proteins in flow-through mode: Purification of protein therapeutics and proteomic study}, volume={326}, ISSN={["1873-3794"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85169830823&partnerID=MN8TOARS}, DOI={10.1016/j.seppur.2023.124777}, abstractNote={K. phaffii is a versatile expression system that is increasingly utilized to produce biological therapeutics – including enzymes, engineered antibodies, and gene-editing tools – that feature multiple subunits and complex post-translational modifications. Two major roadblocks limit the adoption of K. phaffii in industrial biomanufacturing: its proteome, while known, has not been linked to downstream process operations and detailed knowledge is missing on problematic host cell proteins (HCPs) that endanger patient safety or product stability. Furthermore, the purification toolbox has not evolved beyond the capture of monospecific antibodies, and few solutions are available for engineered antibody fragments and other protein therapeutics. To unlock the potential of yeast-based biopharmaceutical manufacturing, this study presents the development and performance validation of a novel adsorbent – PichiaGuard – functionalized with peptide ligands that target the whole spectrum of K. phaffii HCPs and designed for protein purification in flow-through mode. The PichiaGuard adsorbent features high HCP binding capacity (∼25 g per liter of resin) and successfully purified a monoclonal antibody and an ScFv fragment from clarified K. phaffii harvests, affording > 300-fold removal of HCPs and high product yields (70–80%). Notably, PichiaGuard outperformed commercial ion exchange and mixed-mode resins without salt gradients or optimization in removing high-risk HCPs – including aspartic proteases, ribosomal subunits, and other peptidases – thus demonstrating its value in modern biopharmaceutical processing.}, journal={SEPARATION AND PURIFICATION TECHNOLOGY}, author={Sripada, Sobhana A. and Elhanafi, Driss and Collins, Leonard B. and Williams, Taufika I. and Linova, Marina Y. and Woodley, John M. and Boi, Cristiana and Menegatti, Stefano}, year={2023}, month={Dec} }
 @article{chu_prodromou_moore_elhanafi_kilgore_shastry_menegatti_2022, title={Development of peptide ligands for the purification of a-1 antitrypsin from cell culture fluids}, volume={1679}, ISSN={["1873-3778"]}, DOI={10.1016/j.chroma.2022.463363}, abstractNote={α-1 antitrypsin (AAT) deficiency, a major risk factor for chronic obstructive pulmonary disease, is one of the most prevalent and fatal hereditary diseases. The rising demand of AAT poses a defined need for new processes of AAT manufacturing from recombinant sources. Commercial affinity adsorbents for AAT purification present the intrinsic limitations of protein ligands - chiefly, the high cost and the lability towards the proteases in the feedstocks and the cleaning-in-place utilized in biomanufacturing - which limit their application despite their high capacity and selectivity. This work presents the development of small peptide affinity ligands for the purification of AAT from Chinese hamster ovary (CHO) cell culture harvests. An ensemble of ligand candidates identified via library screening were conjugated on Toyopearl resin and evaluated via experimental and in silico AAT-binding studies. Initial ranking based on equilibrium binding capacity indicated WHAKKSKFG- (12.9 mg of AAT per mL of resin), WHAKKSHFG- (16.3 mg/mL), and KWKHSHKWG- (15.8 mg/mL) Toyopearl resins as top performing adsorbents. Notably, the fitting of adsorption data to Langmuir isotherms concurred with molecular docking and dynamics in returning values of dissociation constant (KD) between 1 - 10 µM. These peptide-based adsorbents were thus selected for AAT purification from CHO fluids, affording values of AAT binding capacity up to 13 gram per liter of resin, and product yield and purity up to 77% and 97%. WHAKKSHFG-Toyopearl resin maintained its purification activity upon 20 consecutive uses, demonstrating its potential for AAT manufacturing from recombinant sources.}, journal={JOURNAL OF CHROMATOGRAPHY A}, author={Chu, Wenning and Prodromou, Raphael and Moore, Brandyn and Elhanafi, Driss and Kilgore, Ryan and Shastry, Shriarjun and Menegatti, Stefano}, year={2022}, month={Aug} }
 @article{parsons_azizoglu_elhanafi_kathariou_2021, title={Mutant Construction and Integration Vector-Mediated Genetic Complementation in Listeria monocytogenes}, volume={2220}, ISBN={["978-1-0716-0981-1"]}, ISSN={["1940-6029"]}, DOI={10.1007/978-1-0716-0982-8_14}, abstractNote={Genes that play a role in stress response mechanisms and other phenotypes of Listeria monocytogenes can be identified by construction and screening of mutant libraries. In this chapter, we describe the construction and screening of mutant libraries of L. monocytogenes using the plasmid pMC38, carrying a mariner-based transposon system (TC1/mariner) and constructed by Cao et al. (Appl Environ Microbiol 73:2758–2761, 2007). Following screening of mutant libraries, putative mutants are identified and the transposon is localized, leading to identification of the genes responsible for the phenotype of interest. To confirm the role of the transposon-harboring gene in the relevant phenotype, transposon mutants are genetically complemented with the wild-type gene using the site-specific temperature-sensitive integration vector pPL2, constructed by Lauer et al. (J Bacteriol 184:4177–4186, 2002).}, journal={LISTERIA MONOCYTOGENES, 2 EDITION}, author={Parsons, Cameron and Azizoglu, Reha and Elhanafi, Driss and Kathariou, Sophia}, year={2021}, pages={177–185} }
 @article{dutta_elhanafi_osborne_martinez_kathariou_2014, title={Genetic Characterization of Plasmid-Associated Triphenylmethane Reductase in Listeria monocytogenes}, volume={80}, ISSN={["1098-5336"]}, DOI={10.1128/aem.01398-14}, abstractNote={ABSTRACT}, number={17}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Dutta, Vikrant and Elhanafi, Driss and Osborne, Jason and Martinez, Mira Rakic and Kathariou, Sophia}, year={2014}, month={Sep}, pages={5379–5385} }
 @article{dutta_elhanafi_kathariou_2013, title={Conservation and Distribution of the Benzalkonium Chloride Resistance Cassette bcrABC in Listeria monocytogenes}, volume={79}, ISSN={["1098-5336"]}, DOI={10.1128/aem.01751-13}, abstractNote={ABSTRACT}, number={19}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Dutta, Vikrant and Elhanafi, Driss and Kathariou, Sophia}, year={2013}, month={Oct}, pages={6067–6074} }
 @article{katharios-lanwermeyer_rakic-martinez_elhanafi_ratani_tiedje_kathariou_2012, title={Coselection of Cadmium and Benzalkonium Chloride Resistance in Conjugative Transfers from Nonpathogenic Listeria spp. to Other Listeriae}, volume={78}, ISSN={["0099-2240"]}, DOI={10.1128/aem.02245-12}, abstractNote={ABSTRACT}, number={21}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Katharios-Lanwermeyer, S. and Rakic-Martinez, M. and Elhanafi, D. and Ratani, S. and Tiedje, J. M. and Kathariou, S.}, year={2012}, month={Nov}, pages={7549–7556} }
 @article{yildirim_elhanafi_lin_hitchins_siletzky_kathariou_2010, title={Conservation of Genomic Localization and Sequence Content of Sau3AI-Like Restriction-Modification Gene Cassettes among Listeria monocytogenes Epidemic Clone I and Selected Strains of Serotype 1/2a}, volume={76}, ISSN={["1098-5336"]}, DOI={10.1128/aem.00648-10}, abstractNote={ABSTRACT}, number={16}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Yildirim, Suleyman and Elhanafi, Driss and Lin, Wen and Hitchins, Anthony D. and Siletzky, Robin M. and Kathariou, S.}, year={2010}, month={Aug}, pages={5577–5584} }
 @article{elhanafi_dutta_kathariou_2010, title={Genetic Characterization of Plasmid-Associated Benzalkonium Chloride Resistance Determinants in a Listeria monocytogenes Strain from the 1998-1999 Outbreak}, volume={76}, ISSN={["1098-5336"]}, DOI={10.1128/aem.02056-10}, abstractNote={ABSTRACT}, number={24}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Elhanafi, Driss and Dutta, Vikrant and Kathariou, Sophia}, year={2010}, month={Dec}, pages={8231–8238} }
 @article{chan_elhanafi_kathariou_2008, title={Genomic evidence for interspecies acquisition of chromosomal DNA from Campylobacter jejuni by Campylobacter coli strains of a turkey-associated clonal group (cluster II)}, volume={5}, ISSN={["1556-7125"]}, DOI={10.1089/fpd.2008.0113}, abstractNote={Previous multilocus sequence typing studies of Campylobacter coli from meat animals identified an unusual cluster of strains, primarily from turkeys, termed "cluster II" and characterized by the presence of the C. jejuni aspA103 allele. To characterize the extent of genomic input from C. jejuni in the aspA region of cluster II C. coli, we sequenced the 6.1 kb genomic region upstream of and including aspA from two turkey-derived cluster II strains (C. coli 6979 and C. coli 7474, of ST-1150 and ST-1161, respectively), as well as from a turkey-derived multidrug-resistant strain (C. coli 6818) representing a major sequence type (ST-1101) outside of cluster II. A gene encoding a putative CRP-family transcriptional regulator (CCO0137) was present in C. coli 6818 and the reference strain C. coli RM2228, whose genome has been sequenced, but not in either cluster II strain evaluated. This gene was also absent from C. jejuni NCTC 11168 and C. jejuni RM1221. Moreover, single nucleotide polymorphism (SNP) analysis revealed that in both cluster II strains, genes encoding subunit II of cytochrome d ubiquinol oxidase (cydB) and a putative aspartate racemase (Cj0085c) harbored numerous C. jejuni-specific SNPs. Interestingly, genes encoding subunit I of cytochrome d ubiquinol oxidase (cydA), uracil-DNA glycosylase (ung), and aspartate ammonia-lyase (aspA) harbored C. coli-specific SNPs in certain portions but C. jejuni-specific SNPs in others, suggesting that these were hybrid genes with C. jejuni-derived segments. Analysis of a ung mutant in C. coli 7474 indicated that the putative hybrid ung of this cluster II strain was functional. Our data suggest the occurrence of recombination events that resulted in genomic import of DNA from C. jejuni in the region between cydA and aspA in cluster II strains of C. coli.}, number={4}, journal={FOODBORNE PATHOGENS AND DISEASE}, author={Chan, Kamfai and Elhanafi, Driss and Kathariou, Sophia}, year={2008}, month={Aug}, pages={387–398} }
 @article{cheng_yue_elhanafi_kathariou_2007, title={Absence of serotype-specific surface antigen in laboratory variants of epidemic-associated Listeria monocytogenes strains}, volume={73}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.00473-07}, abstractNote={ABSTRACT}, number={19}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Cheng, Ying and Yue, Lili and Elhanafi, Driss and Kathariou, S.}, year={2007}, month={Oct}, pages={6313–6316} }
 @article{drake_elhanafi_bang_drake_green_jaykus_2006, title={Validation of a green fluorescent protein-labeled strain of Vibrio vulnificus for use in the evaluation of postharvest strategies for handling of raw oysters}, volume={72}, ISSN={["0099-2240"]}, DOI={10.1128/AEM.01091-06}, abstractNote={ABSTRACT}, number={11}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Drake, S. L. and Elhanafi, D. and Bang, W. and Drake, M. A. and Green, D. P. and Jaykus, L. A.}, year={2006}, month={Nov}, pages={7205–7211} }
 @article{johnston_elhanafi_drake_jaykus_2005, title={A simple method for the direct detection of Salmonella and Escherichia coli O157 : H7 from raw alfalfa sprouts and spent irrigation water using PCR}, volume={68}, ISSN={["1944-9097"]}, DOI={10.4315/0362-028X-68.11.2256}, abstractNote={The U.S. Food and Drug Administration recognizes that raw seed sprouts are an important cause of foodborne disease and is now recommending that either spent irrigation water or final product be screened for Salmonella and Escherichia coli O157:H7 as a means of assuring the safety of product intended for consumption. In an effort to streamline such testing efforts, a simple method to preconcentrate pathogens from sprouts and spent irrigation water was investigated to facilitate the direct (without prior cultural enrichment) detection of pathogens using the PCR technique. Alfalfa sprouts and spent irrigation water were seeded with Salmonella enterica serovar Typhimurium and E. coli O157:H7 at 10(-1) to 106 CFU/g or CFU/ml, respectively. Samples were blended (sprouts only) and then centrifuged at high speed to sediment the total bacterial population. The precipitate was processed for DNA isolation, PCR amplification, and amplicon confirmation by Southern hybridization. Mean pathogen recoveries after centrifugation ranged from 96 to 99% for both pathogens in both matrices. Using primers targeting the invA gene for Salmonella Typhimurium and the stx genes of E. coli O157:H7, it was possible to detect both pathogens in alfalfa sprouts at seeding concentrations as low as 10 CFU/g. PCR detection limits for both pathogens from spent irrigation water were 10(-1) CFU/ml, the equivalent of 100 CFU/liter of water. Because spent irrigation water is constitutionally simple, it is particularly well suited for bacterial concentration by simple centrifugation steps. In this study, progress was made toward development of a rapid, inexpensive, and sensitive method for the detection of sprout-associated pathogens that is relevant to current industrial practices and needs.}, number={11}, journal={JOURNAL OF FOOD PROTECTION}, author={Johnston, LM and Elhanafi, D and Drake, M and Jaykus, LA}, year={2005}, month={Nov}, pages={2256–2263} }
 @article{taylor_elhanafi_drake_jaykus_2005, title={Effect of food matrix and cell growth on PCR-based detection of Escherichia coli O157 : H7 in ground beef}, volume={68}, ISSN={["1944-9097"]}, DOI={10.4315/0362-028X-68.2.225}, abstractNote={The purpose of this work was (i) to investigate the feasibility of a previously reported upstream processing method for PCR template preparation to facilitate the detection of Escherichia coli O157:H7 from ground beef and (ii) to assess the impact of cell growth (no growth in the matrix versus growth in the matrix) on molecular detection limits. Two food matrices (autoclaved and raw ground beef) were evaluated in all studies. For no-growth experiments, 10-g meat samples were inoculated with 10(2) to 10(7) CFU/g E. coli O157:H7 and then homogenized. The homogenates were processed to remove large particulates and inhibitors using a two-phase upstream processing method consisting of two sequential centrifugation steps, the second of which used titanous hydroxide to facilitate bacterial immobilization. After upstream processing, sample concentrates were extracted for DNA isolation and amplified by PCR. For growth experiments, 10-g meat samples were inoculated at 1 CFU of E. coli O157:H7 per gram, allowed to grow to 10(2) to 10(7) CFU/g, and then processed for PCR assay. Cell recoveries after upstream processing ranged from 15.9 to 77.6% and were not facilitated by the use of titanous hydroxide, as compared with a saline control (P > 0.05). Bacterial cell recovery and PCR detection limits were similar when comparing autoclaved ground beef and raw ground beef, but cell recoveries were highly variable for raw ground beef samples in which E. coli O157:H7 cells were allowed to grow before processing for detection. Overall, PCR detection limits approximated 10(3) CFU/g of ground beef for all treatments. These results indicate that use of model food systems may not always provide an accurate replication of real-world conditions when evaluating PCR detection limits.}, number={2}, journal={JOURNAL OF FOOD PROTECTION}, author={Taylor, TM and Elhanafi, D and Drake, M and Jaykus, LA}, year={2005}, month={Feb}, pages={225–232} }
 @article{elhanafi_leenanon_bang_drake_2004, title={Impact of cold and cold-acid stress on poststress tolerance and virulence factor expression of Escherichia coli O157: H7}, volume={67}, DOI={10.4315/0362-028X-67.1.19}, abstractNote={The effect of extended cold or cold-acid storage of Escherichia coli O157:H7 on subsequent acid tolerance, freeze-thaw survival, heat tolerance, and virulence factor (Shiga toxin, intimin, and hemolysin) expression was determined. Three E. coli O157:H7 strains were stressed at 4 degrees C in TSB or pH 5.5 TSB for 4 weeks. The acid (TSB [pH 2.0] or simulated gastric fluid [pH 1.5]) tolerance, freeze-thaw (-20 degrees C to 21 degrees C) survival, and heat (56 degrees C) tolerance of stressed cells were compared with those of control cells. The beta-galactosidase activities of stressed and control cells containing a lacZ gene fusion in the stx2, eaeA, or hlyA gene were determined following stress in TSB or pH 5.5 TSB at 37 degrees C and in the exponential and stationary phases. Cold and cold-acid stresses decreased acid tolerance (P < 0.05), with a larger decrease in acid tolerance being observed after cold stress than after cold-acid stress (P < 0.05). Cold stress increased freeze-thaw survival for all three strains (P < 0.05). Prior cold or cold-acid stress had no effect on virulence factor production (P > 0.05), although growth in acidic media (pH 5.5) enhanced eaeA and hlyA expression (P < 0.05). These results indicate that the prolonged storage of E. coli O157:H7 at 4 degrees C has substantial effects on freeze-thaw tolerance but does not affect subsequent virulence gene expression.}, number={1}, journal={Journal of Food Protection}, author={Elhanafi, D. and Leenanon, B. and Bang, W. and Drake, M. A.}, year={2004}, pages={19–26} }
 @article{leenanon_elhanafi_drake_2003, title={Acid adaptation and starvation effects on Shiga toxin production by Escherichia coli O157 : H7}, volume={66}, ISSN={["1944-9097"]}, DOI={10.4315/0362-028X-66.6.970}, abstractNote={Reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay (ELISA), and gene fusion were used to determine differences in the expression of stx-II mRNA and the production of mature Stx protein following acid adaptation or starvation of Escherichia coli O157:H7 (ATCC 43895) and an isogenic rpoS mutant (FRIK 816-3) under static conditions and with shaking. The expression of stx-II mRNA in acid-adapted and starved cells was more extensive than that in nonstressed control cells. This effect was more pronounced for the rpoS mutant. Oxygenation (incubation with shaking) increased stx-II mRNA expression for both strains relative to the level of expression obtained with static conditions. ELISA results indicated that Stx production was enhanced more in the rpoS mutant than in its wild-type parent strain and that oxygenation enhanced Stx production for both strains but there were no detectable differences between stressed and nonstressed cells of either strain. The monitoring of the gene product of Stx-II alone with the use of stx-IIAB::lacZ gene fusions confirmed the induction of aeration and the absence of a stress effect for both the wild type and the rpoS mutant. These results indicate that oxygen enhances stx-II mRNA expression and Stx production in E. coli O157:H7. Stress conditions such as acid adaptation and starvation enhance stx-II toxin mRNA levels but do not enhance subsequent Stx toxin production.}, number={6}, journal={JOURNAL OF FOOD PROTECTION}, author={Leenanon, B and Elhanafi, D and Drake, MA}, year={2003}, month={Jun}, pages={970–977} }