@article{lee_brown_tham_danielsson-tham_lopez-valladares_ward_elhanafi_chen_kathariou_2024, title={Draft genome sequences of Listeria monocytogenes strains from human listeriosis in Sweden harboring premature stop codons in the virulence determinant inlA}, volume={10}, ISSN={["2576-098X"]}, url={https://doi.org/10.1128/mra.00464-24}, DOI={10.1128/mra.00464-24}, journal={MICROBIOLOGY RESOURCE ANNOUNCEMENTS}, author={Lee, Sangmi and Brown, Phillip and Tham, Wilhelm and Danielsson-Tham, Marie-Louise and Lopez-Valladares, Gloria and Ward, Todd and Elhanafi, Driss and Chen, Yi and Kathariou, Sophia}, editor={Rasko, DavidEditor}, year={2024}, month={Oct} } @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{kilgore_moore_sripada_chu_shastry_barbieri_hu_tian_petersen_mohammadifar_et al._2024, title={Peptide ligands for the universal purification of exosomes by affinity chromatography}, volume={8}, ISSN={["1097-0290"]}, DOI={10.1002/bit.28821}, abstractNote={Abstract Exosomes are gaining prominence as vectors for drug delivery, vaccination, and regenerative medicine. Owing to their surface biochemistry, which reflects the parent cell membrane, these nanoscale biologics feature low immunogenicity, tunable tissue tropism, and the ability to carry a variety of payloads across biological barriers. The heterogeneity of exosomes' size and composition, however, makes their purification challenging. Traditional techniques, like ultracentrifugation and filtration, afford low product yield and purity, and jeopardizes particle integrity. Affinity chromatography represents an excellent avenue for exosome purification. Yet, current affinity media rely on antibody ligands whose selectivity grants high product purity, but mandates the customization of adsorbents for exosomes with different surface biochemistry while their binding strength imposes elution conditions that may harm product's activity. Addressing these issues, this study introduces the first peptide affinity ligands for the universal purification of exosomes from recombinant feedstocks. The peptides were designed to (1) possess promiscuous biorecognition of exosome markers, without binding process‐related contaminants and (2) elute the product under conditions that safeguard product stability. Selected ligands SNGFKKHI and TAHFKKKH demonstrated the ability to capture of exosomes secreted by 14 cell sources and purified exosomes derived from HEK293, PC3, MM1, U87, and COLO1 cells with yields of up to 80% and up‐to 50‐fold reduction of host cell proteins (HCPs) upon eluting with pH gradient from 7.4 to 10.5, recommended for exosome stability. SNGFKKHI‐Toyopearl resin was finally employed in a two‐step purification process to isolate exosomes from HEK293 cell fluids, affording a yield of 68% and reducing the titer of HCPs to 68 ng/mL. The biomolecular and morphological features of the isolated exosomes were confirmed by analytical chromatography, Western blot analysis, transmission electron microscopy, nanoparticle tracking analysis.}, journal={BIOTECHNOLOGY AND BIOENGINEERING}, author={Kilgore, Ryan E. and Moore, Brandyn D. and Sripada, Sobhana A. and Chu, Wenning and Shastry, Shriarjun and Barbieri, Eduardo and Hu, Shiqi and Tian, Weihua and Petersen, Heidi and Mohammadifar, Mohammad and et al.}, year={2024}, month={Aug} } @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 Listeria monocytogenes can persistently contaminate food processing environments and tolerate sanitizers. Most sequenced strains are from clinical and environmental sources in the contemporary era, with relatively few prior to extensive food processing and sanitizer use. We report the genome sequences of a diverse panel of 83 strains from 1926 to 1964. }, 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 K. phaffii host cell proteins in flow-through mode: Purification of protein therapeutics and proteomic study}, volume={326}, ISSN={1383-5866}, url={http://dx.doi.org/10.1016/j.seppur.2023.124777}, 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}, publisher={Elsevier BV}, 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}, pages={124777} } @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 The enzyme triphenylmethane reductase (TMR) reduces toxic triphenylmethane dyes into colorless, nontoxic derivatives, and TMR-producing microorganisms have been proposed as bioremediation tools. Analysis of the genome of Listeria monocytogenes H7858 (1998-1999 hot dog outbreak) revealed that the plasmid (pLM80) of this strain harboring a gene cassette ( bcrABC ) conferring resistance to benzalkonium chloride (BC) and other quaternary ammonium disinfectants also harbored a gene ( tmr ) highly homologous to TMR-encoding genes from diverse Gram-negative bacteria. The pLM80-associated tmr was located two genes downstream of bcrABC as part of a putative IS 1216 composite transposon. To confirm the role of tmr in triphenylmethane dye detoxification, we introduced various tmr -harboring fragments of pLM80 in a pLM80-cured derivative of strain H7550, from the same outbreak as H7858, and assessed the resistance of the constructs to the triphenylmethane dyes crystal violet (CV) and malachite green. Transcriptional and subcloning data suggest that the regulation of TMR is complex. Constructs harboring fragments spanning bcrABC and tmr were CV resistant, and in such constructs tmr transcription was induced by sublethal levels of either BC or CV. However, constructs harboring only tmr and its upstream intergenic region could also confer resistance to CV, albeit at lower levels. Screening a panel of BC-resistant L. monocytogenes strains revealed that all those harboring bcrABC and adjacent pLM80 sequences, including tmr , were resistant to CV and decolorized this dye. The findings suggest a potential role of TMR as a previously unknown adaptive attribute for environmental persistence of L. monocytogenes . }, 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 Analysis of a panel of 116 Listeria monocytogenes strains of diverse serotypes and sources (clinical, environment of food processing plants, and food) revealed that all but one of the 71 benzalkonium chloride-resistant (BC r ) isolates harbored bcrABC , previously identified on a large plasmid (pLM80) of the 1998-1999 hot dog outbreak strain H7858. In contrast, bcrABC was not detected among BC-susceptible (BC s ) isolates. The bcrABC sequences were highly conserved among strains of different serotypes, but variability was noted in sequences flanking bcrABC . The majority of the BC r isolates had either the pLM80-type of organization of the bcrABC region or appeared to harbor bcrABC on the chromosome, adjacent to novel sequences. Transcription of bcrABC was induced by BC (10 μg/ml) in strains of different serotypes and diverse bcrABC region organization. These findings reveal widespread dissemination of bcrABC across BC r L. monocytogenes strains regardless of serotype and source, while also suggesting possible mechanisms of bcrABC dissemination across L. monocytogenes genomes. }, 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 Resistance to the quaternary ammonium disinfectant benzalkonium chloride (BC) may be an important contributor to the ability of Listeria spp. to persist in the processing plant environment. Although a plasmid-borne disinfectant resistance cassette ( bcrABC ) has been identified in Listeria monocytogenes , horizontal transfer of these genes has not been characterized. Nonpathogenic Listeria spp. such as L. innocua and L. welshimeri are more common than L. monocytogenes in food processing environments and may contribute to the dissemination of disinfectant resistance genes in listeriae, including L. monocytogenes . In this study, we investigated conjugative transfer of resistance to BC and to cadmium from nonpathogenic Listeria spp. to other nonpathogenic listeriae, as well as to L. monocytogenes . BC-resistant L. welshimeri and L. innocua harboring bcrABC , along with the cadmium resistance determinant cadA2 , were able to transfer resistance to other nonpathogenic listeriae as well as to L. monocytogenes of diverse serotypes, including strains from the 2011 cantaloupe outbreak. Transfer among nonpathogenic Listeria spp. was noticeably higher at 25°C than at 37°C, whereas acquisition of resistance by L. monocytogenes was equally efficient at 25 and 37°C. When the nonpathogenic donors were resistant to both BC and cadmium, acquisition of cadmium resistance was an effective surrogate for transfer of resistance to BC, suggesting coselection between these resistance attributes. The results suggest that nonpathogenic Listeria spp. may behave as reservoirs for disinfectant and heavy metal resistance genes for other listeriae, including the pathogenic species L. monocytogenes . }, 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={ABSTRACTListeria monocytogenesis a food-borne pathogen with a clonal population structure and apparently limited gene flow between strains of different lineages. Strains of epidemic clone I (ECI) have been responsible for numerous outbreaks and invariably have DNA that is resistant to digestion by Sau3AI, suggesting methylation of cytosine at GATC sites. A putative restriction-modification (RM) gene cassette has been identified in the genome of the ECI strain F2365 and all other tested ECI strains but is absent from other strains of the same serotype (4b). Homologous RM cassettes have not been reported amongL. monocytogenesisolates of other serotypes. Furthermore, conclusive evidence for the involvement of this RM cassette in the Sau3AI resistance phenotype of ECI strains has been lacking. In this study, we describe a highly conserved RM cassette in certain strains of serotypes 1/2a and 4a that have Sau3AI-resistant DNA. In these strains the RM cassette was in the same genomic location as in the ECI reference strain F2365. The cassette included a gene encoding a putative recombinase, suggesting insertion via site-specific recombination. Deletion of the RM cassette in the ECI strain F2365 and the serotype 1/2a strain A7 rendered the DNA of both strains susceptible to Sau3AI digestion, providing conclusive evidence that the cassette includes a gene required for methylation of cytosine at GATC sites in both strains. The findings suggest that, in addition to its presence in ECI strains, this RM cassette and the accompanying genomic DNA methylation is also encountered among selected strains of other lineages.}, 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 Quaternary ammonium compounds such as benzalkonium chloride (BC) are widely used as disinfectants in both food processing and medical environments. BC-resistant strains of Listeria monocytogenes have been implicated in multistate outbreaks of listeriosis and have been frequently isolated from food processing plants. However, the genetic basis for BC resistance in L. monocytogenes remains poorly understood. In this study, we have characterized a plasmid (pLM80)-associated BC resistance cassette in L. monocytogenes H7550, a strain implicated in the 1998-1999 multistate outbreak involving contaminated hot dogs. The BC resistance cassette ( bcrABC ) restored resistance to BC (MIC, 40 μg/ml) in a plasmid-cured derivative of H7550. All three genes of the cassette were essential for imparting BC resistance. The transcription of H7550 BC resistance genes was increased under sublethal (10 μg/ml) BC exposure and was higher at reduced temperatures (4, 8, or 25°C) than at 37°C. The level of transcription was higher at 10 μg/ml than at 20 or 40 μg/ml. In silico analysis suggested that the BC resistance cassette was harbored by an IS 1216 composite transposon along with other genes whose functions are yet to be determined. The findings from this study will further our understanding of the adaptations of this organism to disinfectants such as BC and may contribute to the elucidation of possible BC resistance dissemination in L. monocytogenes . }, 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 Variants that lacked reactivity with the serotype 4b-specific monoclonal antibody c74.22 and that lost susceptibility to certain Listeria - or serotype 4b-specific phages were identified in the course of genetic studies with serotype 4b Listeria monocytogenes strains H7550 and F2381L (epidemic clones I and II, respectively). Our findings suggest that such variants can become inadvertently established under laboratory conditions and suggest caution in work involving serotype 4b strains and genetic constructs thereof. }, 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 In this paper we describe a biological indicator which can be used to study the behavior of Vibrio vulnificus , an important molluscan shellfish-associated human pathogen. A V. vulnificus ATCC 27562 derivative that expresses green fluorescent protein (GFP) and kanamycin resistance was constructed using conjugation. Strain validation was performed by comparing the GFP-expressing strain ( Vv- GFP) and the wild-type strain ( Vv -WT) with respect to growth characteristics, heat tolerance (45°C), freeze-thaw tolerance (−20 o and −80°C), acid tolerance (pH 5.0, 4.0, and 3.5), cold storage tolerance (5°C), cold adaptation (15°C), and response to starvation. Levels of recovery were evaluated using nonselective medium (tryptic soy agar containing 2% NaCl) with and without sodium pyruvate. The indicator strain was subsequently used to evaluate the survival of V. vulnificus in oysters exposed to organic acids (citric and acetic acids) and various cooling regimens. In most cases, Vv -GFP was comparable to Vv -WT with respect to growth and survival upon exposure to various biological stressors; when differences between the GFP-expressing and parent strains occurred, they usually disappeared when sodium pyruvate was added to media. When V. vulnificus was inoculated into shellstock oysters, the counts dropped 2 log 10 after 11 to 12 days of refrigerated storage, regardless of the way in which the oysters were initially cooled. Steeper population declines after 12 days of refrigerated storage were observed for both iced and refrigerated products than for slowly cooled product and product held under conservative harvest conditions. By the end of the refrigeration storage study (22 days), the counts of Vv -GFP in iced and refrigerated oysters had reached the limit of detection (10 2 CFU/oyster), but slowly cooled oysters and oysters stored under conservative harvest conditions still contained approximately 10 3 and >10 4 CFU V. vulnificus /oyster by day 22, respectively. The Vv -GFP levels in the oyster meat remained stable for up to 24 h when the meat was exposed to acidic conditions at various pH values. Ease of detection and comparability to the wild-type parent make Vv -GFP a good candidate for use in studying the behavior of V. vulnificus upon exposure to sublethal stressors that might be encountered during postharvest handling of molluscan shellfish. }, 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} }