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1.
Microbiol Resour Announc ; 12(9): e0016323, 2023 Sep 19.
Article in English | MEDLINE | ID: mdl-37504519

ABSTRACT

The continued emergence and spread of antimicrobial resistance among pathogenic bacteria are ever-growing threats to health and economy. Here, we report the draft genomes for 45 Enterobacterales clinical isolates, including historical and contemporary drug-resistant organisms, obtained in Pakistan between 1998 and 2016: 5 Serratia, 3 Salmonella, 3 Enterobacter, and 34 Klebsiella.

2.
Microorganisms ; 10(5)2022 Apr 24.
Article in English | MEDLINE | ID: mdl-35630331

ABSTRACT

Traumatic peripheral nerve injuries tend to be more common in younger, working age populations and can lead to long-lasting disability. Peripheral nerves have an impressive capacity to regenerate; however, successful recovery after injury depends on a number of factors including the mechanism and severity of the trauma, the distance from injury to the reinnervation target, connective tissue sheath integrity, and delay between injury and treatment. Even though modern surgical procedures have greatly improved the success rate, many peripheral nerve injuries still culminate in persistent neuropathic pain and incomplete functional recovery. Recent studies in animals suggest that botulinum neurotoxin A (BoNT/A) can accelerate nerve regeneration and improve functional recovery after injury to peripheral nerves. Possible mechanisms of BoNT/A action include activation or proliferation of support cells (Schwann cells, mast cells, and macrophages), increased angiogenesis, and improvement of blood flow to regenerating nerves.

3.
Front Microbiol ; 12: 787726, 2021.
Article in English | MEDLINE | ID: mdl-35222299

ABSTRACT

Most strains of proteolytic group I Clostridium botulinum (G1 C. botulinum) and some strains of Clostridium sporogenes possess genes encoding botulinum neurotoxin (BoNT), a potent neuroparalytic agent. Within G1 C. botulinum, conserved bont gene clusters of three major toxin serotypes (bont/A/B/F) can be found on conjugative plasmids and/or within chromosomal pathogenicity islands. CRISPR-Cas systems enable site-specific targeting of previously encountered mobile genetic elements (MGE) such as plasmids and bacteriophage through the creation of a spacer library complementary to protospacers within the MGEs. To examine whether endogenous CRISPR-Cas systems restrict the transfer of bont gene clusters across strains we conducted a bioinformatic analysis profiling endogenous CRISPR-Cas systems from 241 G1 C. botulinum and C. sporogenes strains. Approximately 6,200 CRISPR spacers were identified across the strains and Type I-B, III-A/B/D cas genes and CRISPR array features were identified in 83% of the strains. Mapping the predicted spacers against the masked strain and RefSeq plasmid dataset identified 56,000 spacer-protospacer matches. While spacers mapped heavily to targets within bont(+) plasmids, no protospacers were identified within the bont gene clusters. These results indicate the toxin is not a direct target of CRISPR-Cas but the plasmids predominantly responsible for its mobilization are. Finally, while the presence of a CRISPR-Cas system did not reliably indicate the presence or absence of a bont gene cluster, comparative genomics across strains indicates they often occupy the same hypervariable loci common to both species, potentially suggesting similar mechanisms are involved in the acquisition and curation of both genomic features.

4.
Microbiol Resour Announc ; 9(20)2020 May 14.
Article in English | MEDLINE | ID: mdl-32409526

ABSTRACT

Infections in immunocompromised patients that are caused by extensively drug-resistant (XDR) Acinetobacter baumannii strains have been increasingly reported worldwide. In particular, carbapenem-resistant A. baumannii strains are a prominent cause of health care-associated infections. Here, we report draft genome assemblies for two clinical XDR A. baumannii isolates obtained from hospitalized patients in Pakistan.

5.
Front Microbiol ; 11: 239, 2020.
Article in English | MEDLINE | ID: mdl-32153532

ABSTRACT

Clostridium botulinum is a Gram-positive, spore-forming anaerobic bacterium that produces botulinum neurotoxin (BoNT). Closing their genomes provides information about their neurotoxin clusters' arrangement(s) and their location (e.g., chromosome or plasmid) which cannot be assessed using draft genomes. Therefore, we tested the use of long-read sequencing (nanopore sequencing) in combination with short-read sequencing to close two toxin-producing strains. These genomes could be used by the Public Health Emergency Preparedness and Response staff during botulism outbreaks. The genomes of two toxin-producing C. botulinum strains, one from an environmental sample (83F_CFSAN034202) and the other from a clinical sample (CDC51232_CFSAN034200) were sequenced using MinION and MiSeq devices. The genomes, including the chromosomes and the plasmids, were closed by a combination of long-read and short-read sequencing. They belonged to different C. botulinum sequence types (STs), with 83F belonging to ST4 and CDC51232 to ST7. A whole genome single nucleotide polymorphism (SNP) analysis clustered these two strains with strains in lineage 2 (e.g., 6CDC297) and 4 (e.g., NCTC2916) from Group I, respectively. These two strains were also bivalent strains with the BoNTB and BoNTA4 clusters located in the larger plasmid for CDC51232, and the BoNTB and BoNTA1 clusters located both in the chromosome for 83F. Overall, this study showed the advantage of combining these two sequencing methods to obtain high quality closed C. botulinum genomes that could be used for SNP phylogenies (source tracking) as well as for fast identification of BoNT clusters and their gene arrangements.

6.
Health Secur ; 17(6): 439-453, 2019.
Article in English | MEDLINE | ID: mdl-31859568

ABSTRACT

We conducted a comprehensive, multiphase laboratory evaluation of the Plague BioThreat Alert® (BTA) test, a lateral flow immunoassay (LFA), for the rapid detection of Yersinia pestis. The study was conducted in 7 phases at 2 sites to assess the performance of the LFA. The limit of detection (LOD) was determined using both a virulent and avirulent strain of Y. pestis, CO99-3015 (105 CFU/ml) and A1122 (104 CFU/ml), respectively. In the other phases, 18 Y. pestis strains, 20 phylogenetic near-neighbor strains, 61 environmental background microorganisms, 26 white powders, and a pooled aerosol sample were also tested. A total of 1,110 LFA test results were obtained, and their analysis indicates that this LFA had a sensitivity of 97.65% and specificity of 96.57%. These performance data are important for accurate interpretation of qualitative results arising from testing suspicious white powders and aerosol samples in the field. Any positive specimen in this assay is considered presumptive positive and should be referred to the Centers for Disease Control and Prevention Laboratory Response Network for additional testing, confirmation, and characterization for an appropriate public health response.


Subject(s)
Bioterrorism/prevention & control , Immunoassay/methods , Plague/prevention & control , Yersinia pestis/isolation & purification , Humans , Sensitivity and Specificity
7.
Health Secur ; 17(4): 334-343, 2019.
Article in English | MEDLINE | ID: mdl-31433282

ABSTRACT

A comprehensive laboratory evaluation of the Tetracore RedLine Alert test, a lateral flow immunoassay (LFA) for the rapid presumptive identification of Bacillus anthracis, was conducted at 2 different test sites. The study evaluated the sensitivity of this assay using 16 diverse strains of B. anthracis grown on sheep blood agar (SBA) plates. In addition, 83 clinically relevant microorganisms were tested to assess the specificity of the RedLine Alert test. The results indicated that the RedLine Alert test for the presumptive identification of B. anthracis is highly robust, specific, and sensitive. RedLine Alert is a rapid test that has applicability for use in a clinical setting for ruling-in or ruling-out nonhemolytic colonies of Bacillus spp. grown on SBA medium as presumptive isolates of B. anthracis.


Subject(s)
Anthrax , Bacillus anthracis/isolation & purification , Diagnostic Tests, Routine , Immunoassay , Animals , Anthrax/diagnosis , Anthrax/microbiology , Humans , Sensitivity and Specificity , Sheep
8.
Microbiol Resour Announc ; 8(30)2019 Jul 25.
Article in English | MEDLINE | ID: mdl-31346012

ABSTRACT

Shigella spp. are the most common cause of dysentery in developing countries and the second leading cause of diarrheal deaths worldwide. Multidrug-resistant (MDR) Shigella spp. are a serious threat to global health. Herein, we report draft genome sequences for three MDR Shigella isolates from Pakistan, two Shigella flexneri isolates and one Shigella sonnei isolate.

9.
Article in English | MEDLINE | ID: mdl-30533938

ABSTRACT

Here we report the genome sequences of two toxin-producing Clostridium botulinum strains, one environmental sample (83F) and one clinical sample (CDC51232). The genomes were closed by a combination of long-read and short-read sequencing. The strains belong to C. botulinum sequence type 4 (ST4) and ST7, respectively.

10.
PLoS One ; 13(6): e0198526, 2018.
Article in English | MEDLINE | ID: mdl-29883490

ABSTRACT

The emergence and dissemination of carbapenemases, bacterial enzymes able to inactivate most ß-lactam antibiotics, in Enterobacteriaceae is of increasing concern. The concurrent spread of resistance against colistin, an antibiotic of last resort, further compounds this challenge further. Whole-genome sequencing (WGS) can play a significant role in the rapid and accurate detection/characterization of existing and emergent resistance determinants, an essential aspect of public health surveillance and response activities to combat the spread of antimicrobial resistant bacteria. In the current study, WGS data was used to characterize the genomic content of antimicrobial resistance genes, including those encoding carbapenemases, in 10 multidrug-resistant Klebsiella pneumoniae isolates from Pakistan. These clinical isolates represented five sequence types: ST11 (n = 3 isolates), ST14 (n = 3), ST15 (n = 1), ST101 (n = 2), and ST307 (n = 1). Resistance profiles against 25 clinically-relevant antimicrobials were determined by broth microdilution; resistant phenotypes were observed for at least 15 of the 25 antibiotics tested in all isolates except one. Specifically, 8/10 isolates were carbapenem-resistant and 7/10 isolates were colistin-resistant. The blaNDM-1 and blaOXA-48 carbapenemase genes were present in 7/10 and 5/10 isolates, respectively; including 2 isolates carrying both genes. No plasmid-mediated determinants for colistin resistance (e.g. mcr) were detected, but disruptions and mutations in chromosomal loci (i.e. mgrB and pmrB) previously reported to confer colistin resistance were observed. A blaOXA-48-carrying IncL/M-type plasmid was found in all blaOXA-48-positive isolates. The application of WGS to molecular epidemiology and surveillance studies, as exemplified here, will provide both a more complete understanding of the global distribution of MDR isolates and a robust surveillance tool useful for detecting emerging threats to public health.


Subject(s)
Anti-Bacterial Agents/pharmacology , Carbapenems/pharmacology , Colistin/pharmacology , Klebsiella Infections/diagnosis , Klebsiella pneumoniae/drug effects , Bacterial Proteins/genetics , DNA Restriction Enzymes/genetics , DNA, Bacterial/chemistry , DNA, Bacterial/isolation & purification , DNA, Bacterial/metabolism , Drug Resistance, Bacterial/genetics , Humans , Klebsiella Infections/microbiology , Klebsiella pneumoniae/genetics , Klebsiella pneumoniae/isolation & purification , Microbial Sensitivity Tests , Molecular Typing , Plasmids/genetics , Plasmids/metabolism , Whole Genome Sequencing
11.
Genome Announc ; 6(26)2018 Jun 28.
Article in English | MEDLINE | ID: mdl-29954889

ABSTRACT

Clostridium botulinum is a strictly anaerobic, Gram-positive, spore-forming bacterium that produces botulinum neurotoxin, a potent and deadly proteinaceous exotoxin. Clostridium botulinum strain CFSAN064329 (62A) produces an A1 serotype/subtype botulinum neurotoxin and is frequently utilized in food challenge and detection studies. We report here the closed genome sequence of Clostridium botulinum strain CFSAN064329 (62A).

12.
Genome Announc ; 5(48)2017 Nov 30.
Article in English | MEDLINE | ID: mdl-29192076

ABSTRACT

Clostridial neurotoxins, including botulinum and tetanus neurotoxins, are among the deadliest known bacterial toxins. Until recently, the horizontal mobility of this toxin gene family appeared to be limited to the genus Clostridium We report here the closed genome sequence of Chryseobacterium piperi, a Gram-negative bacterium containing coding sequences with homology to clostridial neurotoxin family proteins.

13.
mBio ; 8(6)2017 11 14.
Article in English | MEDLINE | ID: mdl-29138303

ABSTRACT

The continued rise and spread of antimicrobial resistance among bacterial pathogens pose a serious challenge to global health. Countering antimicrobial-resistant pathogens requires a multifaceted effort that includes the discovery of novel therapeutic approaches. Here, we establish the capacity of the human CXC chemokines CXCL9 and CXCL10 to kill multidrug-resistant Gram-negative bacteria, including New Delhi metallo-beta-lactamase-1-producing Klebsiella pneumoniae and colistin-resistant members of the family Enterobacteriaceae that harbor the mobile colistin resistance protein MCR-1 and thus possess phosphoethanolamine-modified lipid A. Colistin-resistant K. pneumoniae isolates affected by genetic mutation of the PmrA/PmrB two-component system, a chromosomally encoded regulator of lipopolysaccharide modification, and containing 4-amino-4-deoxy-l-arabinose-modified lipid A were also found to be susceptible to chemokine-mediated antimicrobial activity. However, loss of PhoP/PhoQ autoregulatory control, caused by disruption of the gene encoding the negative regulator MgrB, limited the bactericidal effects of CXCL9 and CXCL10 in a variable, strain-specific manner. Cumulatively, these findings provide mechanistic insight into chemokine-mediated antimicrobial activity, highlight disparities amongst determinants of colistin resistance, and suggest that chemokine-mediated bactericidal effects merit additional investigation as a therapeutic avenue for treating infections caused by multidrug-resistant pathogens.IMPORTANCE As bacterial pathogens become resistant to multiple antibiotics, the infections they cause become increasingly difficult to treat. Carbapenem antibiotics provide an essential clinical barrier against multidrug-resistant bacteria; however, the dissemination of bacterial enzymes capable of inactivating carbapenems threatens the utility of these important antibiotics. Compounding this concern is the global spread of bacteria invulnerable to colistin, a polymyxin antibiotic considered to be a last line of defense against carbapenem-resistant pathogens. As the effectiveness of existing antibiotics erodes, it is critical to develop innovative antimicrobial therapies. To this end, we demonstrate that the chemokines CXCL9 and CXCL10 kill the most concerning carbapenem- and colistin-resistant pathogens. Our findings provide a unique and timely foundation for therapeutic strategies capable of countering antibiotic-resistant "superbugs."


Subject(s)
Anti-Bacterial Agents/metabolism , Chemokine CXCL10/metabolism , Chemokine CXCL9/metabolism , Enterobacteriaceae/drug effects , Microbial Viability/drug effects , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Enterobacteriaceae/physiology , Humans
14.
Genome Announc ; 4(6)2016 Dec 15.
Article in English | MEDLINE | ID: mdl-27979956

ABSTRACT

The emergence and spread of colistin resistance among multidrug-resistant (MDR) Klebsiella pneumoniae represent a critical threat to global health. Here, we report the complete genome sequences of 10 MDR, colistin-susceptible and -resistant K. pneumoniae clinical isolates obtained in Pakistan between 2010 and 2013.

15.
Health Secur ; 13(1): 37-44, 2015.
Article in English | MEDLINE | ID: mdl-25812427

ABSTRACT

The mouse bioassay (MBA) is the only accepted standard method for detection of botulinum neurotoxins (BoNTs) in foods. The ELISA method has several advantages over the MBA and is therefore widely used for in vitro detection of BoNTs. The US Food and Drug Administration (FDA) and the Centers for Disease Control and Prevention (CDC) conducted a precollaborative study to evaluate the applicability of Botulinum Toxin ELISA kits for the detection of BoNT serotypes A, B, E, and F in a variety of food matrices. In this study, food samples (e.g., broccoli, salami, smoked salmon, green beans, orange juice, tomato juice, low-fat plain yogurt, whole milk, liquid infant formula milk, and liquid eggs) were spiked with high, medium, and low concentration BoNT serotypes A, B, E, and F. Samples (unspiked and spiked) were tested at both laboratories by the ELISA kits. All food samples were positive for BoNTs by ELISA in both laboratories at medium and high spiking levels; a positive ELISA result in low spiked samples was both serotype and laboratory dependent. Overall, the ELISA method appears to be an effective preliminary screening method for BoNT detection in food matrices.


Subject(s)
Botulinum Toxins/analysis , Enzyme-Linked Immunosorbent Assay , Food Microbiology/methods , Food , Animals , Botulinum Toxins, Type A/analysis , Clostridium botulinum/isolation & purification , Mice , Sensitivity and Specificity
16.
Genome Announc ; 2(6)2014 Dec 11.
Article in English | MEDLINE | ID: mdl-25502671

ABSTRACT

Here we report the genome sequence of a Clostridium botulinum strain IBCA10-7060 producing botulinum neurotoxin serotype B and a new toxin serotype. Multilocus sequence typing analysis revealed that this strain belongs to a new sequence type, and whole-genome single nucleotide polymorphism analysis showed that this strain clustered with strains in lineage 2 from group I.

17.
Biosecur Bioterror ; 12(1): 49-62, 2014.
Article in English | MEDLINE | ID: mdl-24552362

ABSTRACT

Abrin is a heterodimeric toxin present in the seeds of the Abrus precatorius plant. The easily obtainable seeds can yield a highly toxic product that can be used in various types of biocrimes and terrorism-related activities, including "white-powder" letters. Although the vast majority of these threats are hoaxes, the lack of rapid and reliable detection assays for abrin, such as lateral flow assays (LFAs), can be an impediment to accurate and rapid hazard assessment. One of the complicating factors associated with LFAs is the use of antibodies of poor affinity and specificity that cross-react with near neighbors or that bind to plant lectins, which are capable of nonspecifically cross-linking the capture and detector antibodies. Because of the critical need to promote public safety and public health, we conducted a comprehensive laboratory evaluation of a commercial LFA for the rapid detection of abrin. This study was conducted using comprehensive inclusivity and exclusivity panels of abrin and near-neighbor plant materials, along with panels of lectins, related proteins, white powders, and environmental background material, to determine the sensitivity, specificity, limit of detection, dynamic range, and repeatability of the assay for the specific intended use of evaluating suspicious white powders and environmental samples for the presumptive presence of abrin.


Subject(s)
Abrin/isolation & purification , Powders/chemistry , Reagent Kits, Diagnostic/standards , Chemical Terrorism , Powders/poisoning , Reagent Strips , Sensitivity and Specificity , United States
18.
Appl Environ Microbiol ; 80(7): 2125-32, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24463972

ABSTRACT

Clostridium botulinum is a genetically diverse Gram-positive bacterium producing extremely potent neurotoxins (botulinum neurotoxins A through G [BoNT/A-G]). The complete genome sequences of three strains harboring only the BoNT/A1 nucleotide sequence are publicly available. Although these strains contain a toxin cluster (HA(+) OrfX(-)) associated with hemagglutinin genes, little is known about the genomes of subtype A1 strains (termed HA(-) OrfX(+)) that lack hemagglutinin genes in the toxin gene cluster. We sequenced the genomes of three BoNT/A1-producing C. botulinum strains: two strains with the HA(+) OrfX(-) cluster (69A and 32A) and one strain with the HA(-) OrfX(+) cluster (CDC297). Whole-genome phylogenic single-nucleotide-polymorphism (SNP) analysis of these strains along with other publicly available C. botulinum group I strains revealed five distinct lineages. Strains 69A and 32A clustered with the C. botulinum type A1 Hall group, and strain CDC297 clustered with the C. botulinum type Ba4 strain 657. This study reports the use of whole-genome SNP sequence analysis for discrimination of C. botulinum group I strains and demonstrates the utility of this analysis in quickly differentiating C. botulinum strains harboring identical toxin gene subtypes. This analysis further supports previous work showing that strains CDC297 and 657 likely evolved from a common ancestor and independently acquired separate BoNT/A1 toxin gene clusters at distinct genomic locations.


Subject(s)
Bacteriological Techniques/methods , Clostridium botulinum/classification , Clostridium botulinum/genetics , Genome, Bacterial , Polymorphism, Single Nucleotide , Cluster Analysis , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Genotype , Molecular Diagnostic Techniques/methods , Molecular Sequence Data , Sequence Analysis, DNA
19.
J Sci Food Agric ; 94(4): 707-12, 2014 Mar 15.
Article in English | MEDLINE | ID: mdl-23873138

ABSTRACT

BACKGROUND: Specific screening methods for complex food matrices are needed that enable unambiguous and sensitive detection of bio threat agents (BTAs) such as Bacillus anthracis spores and microbial toxins (e.g. staphylococcal enterotoxin B (SEB) and clostridial botulinum neurotoxins (BoNTs)). The present study describes an image-based 96-well Meso Scale Discovery (MSD) electrochemiluminescence (ECL) assay for simultaneous detection of BTAs in dairy milk products. RESULTS: The limit of detection of this ECL assay is 40 pg mL⁻¹ for BoNT/A complex, 10 pg mL⁻¹ for SEB and 40000 CFU mL⁻¹ for Bacillus anthracis spores in dairy milk products. The ECL assay was successfully applied to screen type A Clostridium botulinum outbreak strains. CONCLUSION: The results of the study indicate that this ECL assay is very sensitive, rapid (<6 h) and multiplex in nature. The ECL assay has potential for use as an in vitro screening method for BTAs over other comparable immunoassays.


Subject(s)
Bacterial Toxins/analysis , Clostridium botulinum type A/isolation & purification , Dairy Products/analysis , Food Contamination , Food Inspection/methods , Foodborne Diseases/prevention & control , Luminescence , Antigens, Bacterial/analysis , Antigens, Bacterial/chemistry , Antigens, Bacterial/metabolism , Bacillus anthracis/growth & development , Bacillus anthracis/isolation & purification , Bacillus anthracis/physiology , Bacterial Toxins/chemistry , Bacterial Toxins/metabolism , Botulinum Toxins, Type A/analysis , Botulinum Toxins, Type A/chemistry , Botulinum Toxins, Type A/metabolism , Botulism/epidemiology , Botulism/microbiology , Botulism/prevention & control , Clostridium botulinum type A/growth & development , Clostridium botulinum type A/metabolism , Colony Count, Microbial , Dairy Products/adverse effects , Dairy Products/microbiology , Disease Outbreaks/prevention & control , Electrochemical Techniques , Enterotoxins/analysis , Enterotoxins/chemistry , Enterotoxins/metabolism , Food Microbiology , Foodborne Diseases/etiology , Foodborne Diseases/microbiology , Humans , Limit of Detection , Luminescent Measurements , Spores, Bacterial/isolation & purification , United States , United States Food and Drug Administration
20.
Biosecur Bioterror ; 11(4): 237-50, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24320219

ABSTRACT

Ricin, a heterodimeric toxin that is present in the seeds of the Ricinus communis plant, is the biothreat agent most frequently encountered by law enforcement agencies in the United States. Even in untrained hands, the easily obtainable seeds can yield a highly toxic product that has been used in various types of threats, including "white-powder" letters. Although the vast majority of these threats are hoaxes, an impediment to accurate hazard assessments by first responders is the unreliability of rapid detection assays for ricin, such as lateral flow assays (LFAs). One of the complicating factors associated with LFAs is the incorporation of antibodies of poor specificity that cross-react with near-neighbors or with plant lectins that are capable of nonspecifically cross-linking the capture and detector antibodies. Because of the compelling and critical need to promote the interests of public safety and public health, the Department of Homeland Security conducted a comprehensive laboratory evaluation study of a commercial LFA for the rapid detection of ricin. This study was conducted using comprehensive inclusivity and exclusivity panels of ricin and near-neighbor plant materials, along with panels of lectins and "white-powders," to determine the specificity, sensitivity, limits of detection, dynamic range, and repeatability of the assay for the specific intended use of evaluating suspicious white powders and environmental samples in the field.


Subject(s)
Chemical Warfare Agents/analysis , Chromatography, Affinity/methods , Ricin/analysis , Air Filters , Environment , Humans , Laboratories , Limit of Detection , Plant Extracts/analysis , Plant Lectins/analysis , Powders/chemistry , Reproducibility of Results
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