Simultaneous Rapid Detection and Serotyping of Cronobacter sakazakii Serotypes O1, O2, and O3 by Using Specific Monoclonal Antibodies

ABSTRACTCronobacter sakazakiiis a foodborne pathogen associated with rare but often lethal infections in neonates. Powdered infant formula (PIF) represents the most frequent source of infection. Out of the identified serotypes (O1 to O7), O1, O2, and O3 are often isolated from clinical and PIF samples. Serotype-specific monoclonal antibodies (MAbs) suitable for application in enzyme immunoassays (EIAs) for the rapid detection ofC. sakazakiihave not yet been developed. In this study, we created specific MAbs with the ability to bind toC. sakazakiiof serotypes O1, O2, and O3. Characterization by indirect EIAs, immunofluorescence, motility assays, and immunoblotting identified lipopolysaccharide (LPS) and exopolysaccharide (EPS) as the antigenic determinants of the MAbs. The established sandwich EIAs were highly sensitive and were able to detect between 2 × 103and 9 × 106CFU/ml. Inclusivity tests confirmed that 93% of serotype O1 strains, 100% of O2 strains, and 87% of O3 strains were detected at low cell counts. No cross-reactivity with >100 strains ofCronobacterspp. and otherEnterobacteriaceaewas observed, except for that withC. sakazakiiserotype O3 andCronobactermuytjensiiserotype O1. Moreover, the sandwich EIAs detectedC. sakazakiiin PIF samples artificially contaminated with 1 to 10 bacterial cells per 10 g of sample after 15 h of preenrichment. The use of these serotype-specific MAbs not only allows the reliable detection ofC. sakazakiistrains but also enables simultaneous serotyping in a simple sandwich EIA method.

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Characterization of components of Z-bands in the fibrillar flight muscle of Drosophila melanogaster.

The Journal of Cell Biology ◽

10.1083/jcb.109.5.2157 ◽

1989 ◽

Vol 109 (5) ◽

pp. 2157-2167 ◽

Cited By ~ 78

Author(s):

J D Saide ◽

S Chin-Bow ◽

J Hogan-Sheldon ◽

L Busquets-Turner ◽

J O Vigoreaux ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Monoclonal Antibodies ◽

Flight Muscle ◽

Cross Reactivity ◽

Antigenic Determinants ◽

Immunogold Staining ◽

Thick Filaments ◽

The Matrix ◽

Flight Muscles

Twelve monoclonal antibodies have been raised against proteins in preparations of Z-disks isolated from Drosophila melanogaster flight muscle. The monoclonal antibodies that recognized Z-band components were identified by immunofluorescence microscopy of flight muscle myofibrils. These antibodies have identified three Z-disk antigens on immunoblots of myofibrillar proteins. Monoclonal antibodies alpha:1-4 recognize a 90-100-kD protein which we identify as alpha-actinin on the basis of cross-reactivity with antibodies raised against honeybee and vertebrate alpha-actinins. Monoclonal antibodies P:1-4 bind to the high molecular mass protein, projectin, a component of connecting filaments that link the ends of thick filaments to the Z-band in insect asynchronous flight muscles. The anti-projectin antibodies also stain synchronous muscle, but, surprisingly, the epitopes here are within the A-bands, not between the A- and Z-bands, as in flight muscle. Monoclonal antibodies Z(210):1-4 recognize a 210-kD protein that has not been previously shown to be a Z-band structural component. A fourth antigen, resolved as a doublet (approximately 400/600 kD) on immunoblots of Drosophila fibrillar proteins, is detected by a cross reacting antibody, Z(400):2, raised against a protein in isolated honeybee Z-disks. On Lowicryl sections of asynchronous flight muscle, indirect immunogold staining has localized alpha-actinin and the 210-kD protein throughout the matrix of the Z-band, projectin between the Z- and A-bands, and the 400/600-kD components at the I-band/Z-band junction. Drosophila alpha-actinin, projectin, and the 400/600-kD components share some antigenic determinants with corresponding honeybee proteins, but no honeybee protein interacts with any of the Z(210) antibodies.

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RNA Sequencing-Based Transcriptional Overview of Xerotolerance inCronobacter sakazakiiSP291

Applied and Environmental Microbiology ◽

10.1128/aem.01993-18 ◽

2018 ◽

Vol 85 (3) ◽

Cited By ~ 6

Author(s):

Shabarinath Srikumar ◽

Yu Cao ◽

Qiongqiong Yan ◽

Koenraad Van Hoorde ◽

Scott Nguyen ◽

...

Keyword(s):

Infant Formula ◽

Mortality Rates ◽

Secondary Response ◽

Ecological Niches ◽

Infant Food ◽

Powdered Infant Formula ◽

Cronobacter Sakazakii ◽

Rna Seq ◽

Production Environment ◽

Content Type

ABSTRACTCronobacter sakazakiiis a xerotolerant neonatal pathogen epidemiologically linked to powdered infant food formula, often resulting in high mortality rates. Here, we used transcriptome sequencing (RNA-seq) to provide transcriptional insights into the survival ofC. sakazakiiin desiccated conditions. Our RNA-seq data show that about 22% of the totalC. sakazakiigenes were significantly upregulated and 9% were downregulated during desiccation survival. When reverse transcription-quantitative PCR (qRT-PCR) was used to validate the RNA-seq data, we found that the primary desiccation response was gradually downregulated during the tested 4 hours of desiccation, while the secondary response remained constitutively upregulated. The 4-hour desiccation tolerance ofC. sakazakiiwas dependent on the immediate microenvironment surrounding the bacterial cell. The removal of Trypticase soy broth (TSB) salts and the introduction of sterile infant formula residues in the microenvironment enhanced the desiccation survival ofC. sakazakiiSP291. The trehalose biosynthetic pathway encoded byotsAandotsB, a prominent secondary bacterial desiccation response, was highly upregulated in desiccatedC. sakazakii.C. sakazakiiSP291 ΔotsABwas significantly inhibited compared with the isogenic wild type in an 8-hour desiccation survival assay, confirming the physiological importance of trehalose in desiccation survival. Overall, we provide a comprehensive RNA-seq-based transcriptional overview along with confirmation of the phenotypic importance of trehalose metabolism inCronobacter sakazakiiduring desiccation.IMPORTANCECronobacter sakazakiiis a pathogen of importance to neonatal health and is known to persist in dry food matrices, such as powdered infant formula (PIF) and its associated production environment. When infections are reported in neonates, mortality rates can be high. The success of this bacterium in surviving these low-moisture environments suggests thatCronobacterspecies can respond to a variety of environmental signals. Therefore, understanding those signals that aid the persistence of this pathogen in these ecological niches is an important step toward the development of strategies to reduce the risk of contamination of PIF. This research led to the identification of candidate genes that play a role in the persistence of this pathogen in desiccated conditions and, thereby, serve as a model target to design future strategies to mitigate PIF-associated survival ofC. sakazakii.

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Novel Method for Reliable Identification of Siccibacter and Franconibacter Strains: from “Pseudo-Cronobacter” to New Enterobacteriaceae Genera

Applied and Environmental Microbiology ◽

10.1128/aem.00234-17 ◽

2017 ◽

Vol 83 (13) ◽

Cited By ~ 6

Author(s):

Barbora Svobodová ◽

Jiří Vlach ◽

Petra Junková ◽

Ludmila Karamonová ◽

Martina Blažková ◽

...

Keyword(s):

Intact Cell ◽

Foodborne Pathogen ◽

Principal Component ◽

Powdered Infant Formula ◽

Content Type ◽

Reliable Identification ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Maldi Biotyper ◽

Tof Ms

ABSTRACT In the last decade, strains of the genera Franconibacter and Siccibacter have been misclassified as first Enterobacter and later Cronobacter. Because Cronobacter is a serious foodborne pathogen that affects premature neonates and elderly individuals, such misidentification may not only falsify epidemiological statistics but also lead to tests of powdered infant formula or other foods giving false results. Currently, the main ways of identifying Franconibacter and Siccibacter strains are by biochemical testing or by sequencing of the fusA gene as part of Cronobacter multilocus sequence typing (MLST), but in relation to these strains the former is generally highly difficult and unreliable while the latter remains expensive. To address this, we developed a fast, simple, and most importantly, reliable method for Franconibacter and Siccibacter identification based on intact-cell matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Our method integrates the following steps: data preprocessing using mMass software; principal-component analysis (PCA) for the selection of mass spectrum fingerprints of Franconibacter and Siccibacter strains; optimization of the Biotyper database settings for the creation of main spectrum projections (MSPs). This methodology enabled us to create an in-house MALDI MS database that extends the current MALDI Biotyper database by including Franconibacter and Siccibacter strains. Finally, we verified our approach using seven previously unclassified strains, all of which were correctly identified, thereby validating our method. IMPORTANCE We show that the majority of methods currently used for the identification of Franconibacter and Siccibacter bacteria are not able to properly distinguish these strains from those of Cronobacter. While sequencing of the fusA gene as part of Cronobacter MLST remains the most reliable such method, it is highly expensive and time-consuming. Here, we demonstrate a cost-effective and reliable alternative that correctly distinguishes between Franconibacter, Siccibacter, and Cronobacter bacteria and identifies Franconibacter and Siccibacter at the species level. Using intact-cell MALDI-TOF MS, we extend the current MALDI Biotyper database with 11 Franconibacter and Siccibacter MSPs. In addition, the use of our approach is likely to lead to a more reliable identification scheme for Franconibacter and Siccibacter strains and, consequently, a more trustworthy epidemiological picture of their involvement in disease.

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Idiotypy of clonal responses to influenza virus hemagglutinin.

Journal of Experimental Medicine ◽

10.1084/jem.154.5.1525 ◽

1981 ◽

Vol 154 (5) ◽

pp. 1525-1538 ◽

Cited By ~ 23

Author(s):

Y N Liu ◽

C A Bona ◽

J L Schulman

Keyword(s):

Influenza Virus ◽

Monoclonal Antibodies ◽

Binding Sites ◽

Cross Reactivity ◽

Secondary Response ◽

Antigenic Determinants ◽

Antiviral Responses ◽

Influenza Virus Hemagglutinin ◽

The Cross

Anti-idiotype antisera were raised in syngeneic (BALB/c mice) and homologous (A/J mice) systems to study the cross-reactive idiotypes among monoclonal antibodies to PR8 and B/Lee virus HA and the expression of these idiotypes during primary and secondary antiviral responses of BALB/c mice. Extensive idiotypic cross-reactivity was demonstrated among monoclonal antibodies specific for distinct antigenic determinants on PR8 hemagglutinin (HA). The study of idiotypy of monoclonal antibodies against the same or overlapping antigenic determinants on B/Lee HA showed that these monoclonal antibodies may bear (a) a true individual idiotype not shared by other monoclonal antibodies, (b) idiotypes shared by few monoclonal antibodies, and (c) true cross-reactive idiotypes shared by all of these monoclonal antibodies. In contrast, no cross-reactive idiotypes were detectable among monoclonal antibodies to B/Lee HA and monoclonal antibodies to PR8 HA. Furthermore, we have shown that the anti-idiotype antibodies we used recognize determinants on monoclonal antibodies closely associated with antigenic binding sites. Finally, studies of the idiotypes expressed during primary and secondary antiviral HA responses of mice immunized with B/Lee virus revealed persistence of some idiotypes during both primary and secondary responses, whereas others were only expressed in the primary or secondary response.

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Draft Genome Sequence of Cronobacter sakazakii Cr268, Isolated from Infant Powder Formula

Microbiology Resource Announcements ◽

10.1128/mra.01067-19 ◽

2019 ◽

Vol 8 (46) ◽

Cited By ~ 1

Author(s):

Gal Zizelski Valenci ◽

Mor Rubinstein ◽

Reuven Afriat ◽

Shira Rosencwaig ◽

Zeev Dveyrin ◽

...

Keyword(s):

Infant Formula ◽

Necrotizing Enterocolitis ◽

Genome Sequence ◽

Premature Infants ◽

Food Chain ◽

Draft Genome ◽

Powdered Infant Formula ◽

Cronobacter Sakazakii ◽

Emerging Pathogen ◽

Content Type

Cronobacter sakazakii is an emerging pathogen that causes meningitis, bacteremia, sepsis, and necrotizing enterocolitis in premature infants. Strain Cr268 was isolated from imported powdered infant formula in 2009 during routine microbial examination according to ISO-22964 (“Microbiology of the food chain—horizontal method for the detection of Cronobacter spp.”).

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Production and characterization of monoclonal antibodies to the subunits of human phosphofructokinase: new tools for the immunochemical and genetic analyses of isozymes

Blood ◽

10.1182/blood.v58.4.823.823 ◽

1981 ◽

Vol 58 (4) ◽

pp. 823-829 ◽

Cited By ~ 11

Author(s):

S Vora ◽

LA Wims ◽

S Durham ◽

SL Morrison

Keyword(s):

Monoclonal Antibodies ◽

Protein A ◽

Cross Reactivity ◽

Antigenic Determinants ◽

Vertebrate Species ◽

Muscle Type ◽

Immunoprecipitation Assay ◽

Specific Antibodies ◽

The One

Abstract Recently we have demonstrated that human phosphofructokinase (PFK; ATP: D-fructose-6-P, 1-phosphotransferase; EC.2.7.1.11) is under the control of three structural loci that code for M (muscle-type), L (liver-type), and P (platelet-type) subunits: random tetramerization of these subunits produces various isozymes. In this study, we have produced and characterized BALB/c hybridoma antibodies to the M- and L-type subunits of human PFK. The specific antibodies were detected by an enzyme- immunoprecipitation assay using Staphylococci-bearing protein A as an immunoadsorbent. Of the wells tested using red blood cell (RBC) PFK (M + L), 61% were positive. Only one M-specific hybridoma was identified. The one anti-M and 4 anti-L antibodies were characterized for their biochemical and immunochemical specificities. To define the combining specificities of these antibodies, we compared their reactivity and that of monospecific rabbit anti-M antiserum with muscle and liver PFKs from 15 different vertebrate species. The rabbit anti-M shows strong cross-reactivity with the muscle PFKs from all the species studied. In contrast, the monoclonal anti-M reacts exclusively with muscle PFKs from primates. Two of four anti-L antibodies react only with human L- PFK, whereas the other two react with that from a few other vertebrate species as well. Taken together, these data suggest that primate- specific antibodies recognize evolutionarily, recently acquired antigenic determinants, whereas the antibodies reactive with PFKs from distantly related species recognize conserved determinants. The differential immunoreactivities of muscle and liver PFKs strongly suggest the presence of distinct isozymes in all the vertebrate species studied. These studies demonstrate that it is feasible to produce and characterize monoclonal antibodies that distinguish among isozymes with structural and functional similarities. These antibodies provide sensitive tools in the analyses of isozyme structure, genetics, and related fields.

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Bactericidal Activity of ACH-702 against Nondividing and Biofilm Staphylococci

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00092-12 ◽

2012 ◽

Vol 56 (7) ◽

pp. 3812-3818 ◽

Cited By ~ 12

Author(s):

Steven D. Podos ◽

Jane A. Thanassi ◽

Melissa Leggio ◽

Michael J. Pucci

Keyword(s):

Protein Synthesis ◽

Stationary Phase ◽

Bactericidal Activity ◽

Bacterial Infections ◽

De Novo ◽

Viable Cell ◽

Bacterial Cells ◽

Protein Synthesis Inhibitors ◽

Content Type ◽

Cell Counts

ABSTRACTMany bacterial infections involve slow or nondividing bacterial growth states and localized high cell densities. Antibiotics with demonstrated bactericidal activity rarely remain bactericidal at therapeutic concentrations under these conditions. The isothiazoloquinolone (ITQ) ACH-702 is a potent, bactericidal compound with activity against many antibiotic-resistant pathogens, including methicillin-resistantStaphylococcus aureus(MRSA). We evaluated its bactericidal activity under conditions where bacterial cells were not dividing and/or had slowed their growth. AgainstS. aureuscultures in stationary phase, ACH-702 showed concentration-dependent bactericidal activity and achieved a 3-log-unit reduction in viable cell counts within 6 h of treatment at ≥16× MIC values; in comparison, the bactericidal quinolone moxifloxacin and the additional comparator compounds vancomycin, linezolid, and rifampin at 16× to 32× MICs showed little or no bactericidal activity against stationary-phase cells. ACH-702 at 32× MIC retained bactericidal activity against stationary-phaseS. aureusacross a range of inoculum densities. ACH-702 did not kill cold-arrested cells yet remained bactericidal against cells arrested by protein synthesis inhibitors, suggesting that its bactericidal activity against nondividing cells requires active metabolism but notde novoprotein synthesis. ACH-702 also showed a degree of bactericidal activity at 16× MIC againstS. epidermidisbiofilm cells that was superior to that of moxifloxacin, rifampin, and vancomycin. The bactericidal activity of ACH-702 against stationary-phase staphylococci and biofilms suggests potential clinical utility in infections containing cells in these physiological states.

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High-Sensitivity Monoclonal Antibodies Specific for Homoserine Lactones Protect Mice from Lethal Pseudomonas aeruginosa Infections

Applied and Environmental Microbiology ◽

10.1128/aem.02912-13 ◽

2013 ◽

Vol 80 (2) ◽

pp. 462-469 ◽

Cited By ~ 20

Author(s):

Soumya Palliyil ◽

Christina Downham ◽

Ian Broadbent ◽

Keith Charlton ◽

Andrew J. Porter

Keyword(s):

Pseudomonas Aeruginosa ◽

Monoclonal Antibodies ◽

Bacterial Infections ◽

Recombinant Antibody ◽

Cell Communication ◽

High Sensitivity ◽

Therapeutic Benefit ◽

Cross Reactivity ◽

Content Type ◽

Homoserine Lactones

ABSTRACTA number of bacteria, including pathogens likePseudomonas aeruginosa, utilize homoserine lactones (HSLs) as quorum sensing (QS) signaling compounds and engage in cell-to-cell communication to coordinate their behavior. Blocking this bacterial communication may be an attractive strategy for infection control as QS takes a central role inP. aeruginosabiology. In this study, immunomodulation of HSL molecules by monoclonal antibodies (MAbs) was used as a novel approach to preventP. aeruginosainfections and as tools to detect HSLs in bodily fluids as a possible first clue to an undiagnosed Gram-negative infection. Using sheep immunization and recombinant antibody technology, a panel of sheep-mouse chimeric MAbs were generated which recognized HSL compounds with high sensitivity (nanomolar range) and cross-reactivity. These MAbs retained their nanomolar sensitivity in complex matrices and were able to recognize HSLs inP. aeruginosacultures grown in the presence of urine. In a nematode slow-killing assay, HSL MAbs significantly increased the survival of worms fed on the antibiotic-resistant strain PA058. The therapeutic benefit of these MAbs was further studied using a mouse model ofPseudomonasinfection in which groups of mice treated with HSL-2 and HSL-4 MAbs survived, 7 days after pathogen challenge, in significantly greater numbers (83 and 67%, respectively) compared with the control groups. This body of work has provided early proof-of-concept data to demonstrate the potential of HSL-specific, monoclonal antibodies as theranostic clinical leads suitable for the diagnosis, prevention, and treatment of life-threatening bacterial infections.

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Monoclonal antibodies against human, bovine and rat prolactin: epitope mapping of human prolactin and development of a two-site immunoradiometric assay

Journal of Endocrinology ◽

10.1677/joe.0.1140311 ◽

1987 ◽

Vol 114 (2) ◽

pp. 311-318 ◽

Cited By ~ 7

Author(s):

B. Staindl ◽

P. Berger ◽

R. Kofler ◽

G. Wick

Keyword(s):

Monoclonal Antibodies ◽

Cell Lines ◽

Epitope Mapping ◽

Solid Phase ◽

Cross Reactivity ◽

Placental Lactogen ◽

Antigenic Determinants ◽

Cross Reactions ◽

Human Prolactin ◽

Hybridoma Cell Lines

ABSTRACT Nine mouse hybridoma cell lines producing monoclonal antibodies (MCA) against human prolactin (hPRL), 19 cell lines against bovine prolactin (bPRL) and one MCA against rat prolactin (rPRL) were established. The MCA were characterized by one- and two-site radioimmunoassays (RIA) as well as indirect immunofluorescence (IIF) and used for epitope mapping of hPRL and immunoradiometric assays (IRMA). Interspecies cross-reactivity studies by RIA revealed two groups of anti-hPRL MCA: seven which reacted only with hPRL and two additionally recognizing bPRL and ovine prolactin (oPRL). The anti-bPRL MCA, which were tested on pituitary sections by IIF could be divided into 17 MCA cross-reacting with the closely related oPRL, and two MCA which showed additional cross-reactions with equine prolactin. The anti-rPRL antibody reacted exclusively with rPRL in direct binding RIA studies. No intraspecies cross-reactions with the closely related protein hormones placental lactogen and GH were detected. To elucidate the antigenic surface of hPRL all MCA directed against hPRL were then used for two-site epitope mapping studies in which pairs of MCA were assessed for simultaneous reaction with the same antigen. The native hormone was incubated with the first, solid-phase bound, so called 'capture MCA', and this complex treated with a second, 125I-labelled 'detection MCA'. Based on the results of these combinations, at least three sterically non-overlapping and (taking RIA cross-reaction studies into consideration) two additional epitopes could be defined. Two antibodies (code numbers INN-hPRL-1 and INN-hPRL-9) recognizing different antigenic determinants were selected and used to elaborate a two-site IRMA with an operating range wider and a reaction time shorter than those obtained with a conventional one-site RIA. J. Endocr. (1987) 114,311–318

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