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1.
Int J Mol Sci ; 21(16)2020 Aug 18.
Article in English | MEDLINE | ID: mdl-32824772

ABSTRACT

Infectious diarrhea affects over four billion individuals annually and causes over a million deaths each year. Though not typically prescribed for treatment of uncomplicated diarrheal disease, antimicrobials serve as a critical part of the armamentarium used to treat severe or persistent cases. Due to widespread over- and misuse of antimicrobials, there has been an alarming increase in global resistance, for which a standardized methodology for geographic surveillance would be highly beneficial. To demonstrate that a standardized methodology could be used to provide molecular surveillance of antimicrobial resistance (AMR) genes, we initiated a pilot study to test 130 diarrheal pathogens (Campylobacter spp., Escherichia coli, Salmonella, and Shigella spp.) from the USA, Peru, Egypt, Cambodia, and Kenya for the presence/absence of over 200 AMR determinants. We detected a total of 55 different determinants conferring resistance to ten different categories of antimicrobials: genes detected in ≥ 25 samples included blaTEM, tet(A), tet(B), mac(A), mac(B), aadA1/A2, strA, strB, sul1, sul2, qacEΔ1, cmr, and dfrA1. The number of determinants per strain ranged from none (several Campylobacter spp. strains) to sixteen, with isolates from Egypt harboring a wider variety and greater number of genes per isolate than other sites. Two samples harbored carbapenemase genes, blaOXA-48 or blaNDM. Genes conferring resistance to azithromycin (ere(A), mph(A)/mph(K), erm(B)), a first-line therapeutic for severe diarrhea, were detected in over 10% of all Enterobacteriaceae tested: these included >25% of the Enterobacteriaceae from Egypt and Kenya. Forty-six percent of the Egyptian Enterobacteriaceae harbored genes encoding CTX-M-1 or CTX-M-9 families of extended-spectrum ß-lactamases. Overall, the data provide cross-comparable resistome information to establish regional trends in support of international surveillance activities and potentially guide geospatially informed medical care.


Subject(s)
Campylobacter/genetics , Diarrhea/microbiology , Drug Resistance, Microbial , Enteropathogenic Escherichia coli/genetics , Genes, Bacterial , Salmonella/genetics , Shigella/genetics , Anti-Bacterial Agents/toxicity , Campylobacter/drug effects , Campylobacter/isolation & purification , Campylobacter/pathogenicity , Diarrhea/epidemiology , Enteropathogenic Escherichia coli/drug effects , Enteropathogenic Escherichia coli/isolation & purification , Enteropathogenic Escherichia coli/pathogenicity , Humans , Salmonella/drug effects , Salmonella/isolation & purification , Salmonella/pathogenicity , Shigella/drug effects , Shigella/isolation & purification , Shigella/pathogenicity
2.
Malar J ; 12: 394, 2013 Nov 05.
Article in English | MEDLINE | ID: mdl-24191920

ABSTRACT

BACKGROUND: Experimental vaccines targeting Plasmodium falciparum have had some success in recent years. These vaccines use attenuated parasites, recombinant sporozoite proteins, or DNA and virus combinations to induce cell-mediated immune responses and/or antibodies targeting sporozoite surface proteins. To capitalize on the success of these vaccines and understand the mechanisms by which these vaccines function, it is important to develop assays that measure correlates of protection in volunteers. The inhibition of liver stage development assay (ILSDA) tests antibodies for the ability to block sporozoite development in hepatocytes. As such the ILSDA is an excellent candidate assay to identify correlates of humoral protection, particularly against the liver stage of malaria infection. In addition, the ILSDA can be used as a tool to evaluate novel sporozoite antigens for future vaccine development. Historically the ILSDA has suffered from low sporozoite infection rates, absence of standardized reagents, and the subjectivity associated with the traditional primary outcome measures, which depend on microscopy of stained hepatocyte cultures. This study worked to significantly improve sporozoite infection rates in hepatocytes, modify key steps in the assay protocol to reduce experimental variability, and demonstrate the utility of the ILSDA in testing antibodies targeting the circumsporozoite protein. METHODS: Cryopreserved primary human hepatocytes, Plasmodium falciparum sporozoites, and circumsporozoite antibodies were used to optimize the ILSDA. RESULTS: Inoculation of cryopreserved primary human hepatocytes with Plasmodium falciparum sporozoites improved liver stage development in the ILSDA compared to HCO4 cells. In the ILSDA, circumsporozoite antibodies suppressed liver stage development in cryopreserved primary human hepatocytes in a concentration-dependent manner. Antibody-mediated suppression of parasite development in the ILSDA at a 96-hour endpoint was more robust than the 24-hour endpoint. CONCLUSIONS: ILSDA performance is improved by the use of cryopreserved primary human hepatocytes, expediting interactions between sporozoites and hepatocytes, and extending the assay endpoint.


Subject(s)
Antibodies, Protozoan/immunology , Cell Differentiation/drug effects , Hepatocytes/parasitology , Parasitology/methods , Plasmodium falciparum/immunology , Plasmodium falciparum/physiology , Cell Differentiation/immunology , Cells, Cultured , Cryopreservation , Dose-Response Relationship, Immunologic , Humans , Plasmodium falciparum/drug effects , Plasmodium falciparum/growth & development
4.
Appl Environ Microbiol ; 74(21): 6756-65, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18791020

ABSTRACT

The Sinorhizobium meliloti ORFeome project cloned 6,314 open reading frames (ORFs) into a modified Gateway entry vector system from which the ORFs could be transferred to destination vectors in vivo via bacterial conjugation. In this work, a reporter gene destination vector, pMK2030, was constructed and used to generate ORF-specific transcriptional fusions to beta-glucuronidase (gusA) and green fluorescent protein (gfp) reporter genes. A total of 6,290 ORFs were successfully transferred from the entry vector library into pMK2030. To demonstrate the utility of this system, reporter plasmids corresponding to 30 annotated sugar kinase genes were integrated into the S. meliloti SM1021 and/or SM8530 genome. Expression of these genes was measured using a high-throughput beta-glucuronidase assay to track expression on nine different carbon sources. Six ORFs integrated into SM1021 and SM8530 had different basal levels of expression in the two strains. The annotated activities of three other sugar kinases were also confirmed.


Subject(s)
Artificial Gene Fusion , Bacterial Proteins/metabolism , Glucuronidase/metabolism , Green Fluorescent Proteins/metabolism , Recombinant Fusion Proteins/metabolism , Sinorhizobium meliloti/genetics , Sinorhizobium meliloti/metabolism , Staining and Labeling/methods , Bacterial Proteins/genetics , DNA, Bacterial , Gene Expression Profiling , Genes, Reporter , Genetic Vectors , Glucuronidase/genetics , Green Fluorescent Proteins/genetics , Molecular Sequence Data , Plasmids , Recombinant Fusion Proteins/genetics , Recombination, Genetic , Sequence Analysis, DNA
5.
Afr J Lab Med ; 4(1): 1-7, 2015.
Article in English | MEDLINE | ID: mdl-38440309

ABSTRACT

Background: Food-borne diseases pose serious health problems, affecting public health and economic development worldwide. Methods: Salmonella was isolated from samples of chicken parts, skin samples of whole chicken carcasses, raw egg yolks, eggshells and chicken faeces. Resulting isolates were characterised by serogrouping, serotyping, antimicrobial susceptibility testing and detection of extended-spectrum ß-lactamase (ESBL) production. Antibiotic resistance genes and integrons were identified by polymerase chain reaction (PCR). Results: The detection rates of Salmonella were 60%, 64% and 62% in chicken parts, skin, and faeces, respectively, whereas the egg yolks and eggshells were uniformly negative. Salmonella Kentucky and S. Enteritidis serotypes comprised 43.6% and 2.6% of the isolates, respectively, whilst S. Typhimurium was absent. Variable resistance rates were observed against 16 antibiotics; 97% were resistant to sulfamethoxazole, 96% to nalidixic acid and tetracycline and 76% to ampicillin. Multidrug resistance was detected in 82% (64/78) of the isolates and ESBL production was detected in 8% (6/78). The ß-lactamase blaTEM-1 gene was detected in 57.6% and blaSHV-1 in 6.8% of the isolates, whilst the blaOXA gene was absent. The sul1 gene was detected in 97.3% and the sul2 gene in 5.3% of the isolates. Sixty-four of the 78 isolates (82%) were positive for the integrase gene (int I) from class 1 integrons, whilst int II was absent. Conclusion: This study reveals the presence of an alarming number of multidrug-resistant Salmonella isolates in the local poultry markets in Cairo. The high levels of drug resistance suggest an emerging problem that could impact negatively on efforts to prevent and treat poultry and poultry-transmitted human diseases in Egypt.

6.
PLoS One ; 8(7): e69507, 2013.
Article in English | MEDLINE | ID: mdl-23936031

ABSTRACT

Molecular methods that enable the detection of antimicrobial resistance determinants are critical surveillance tools that are necessary to aid in curbing the spread of antibiotic resistance. In this study, we describe the use of the Antimicrobial Resistance Determinant Microarray (ARDM) that targets 239 unique genes that confer resistance to 12 classes of antimicrobial compounds, quaternary amines and streptothricin for the determination of multidrug resistance (MDR) gene profiles. Fourteen reference MDR strains, which either were genome, sequenced or possessed well characterized drug resistance profiles were used to optimize detection algorithms and threshold criteria to ensure the microarray's effectiveness for unbiased characterization of antimicrobial resistance determinants in MDR strains. The subsequent testing of Acinetobacter baumannii, Escherichia coli and Klebsiella pneumoniae hospital isolates revealed the presence of several antibiotic resistance genes [e.g. belonging to TEM, SHV, OXA and CTX-M classes (and OXA and CTX-M subfamilies) of ß-lactamases] and their assemblages which were confirmed by PCR and DNA sequence analysis. When combined with results from the reference strains, ~25% of the ARDM content was confirmed as effective for representing allelic content from both Gram-positive and -negative species. Taken together, the ARDM identified MDR assemblages containing six to 18 unique resistance genes in each strain tested, demonstrating its utility as a powerful tool for molecular epidemiological investigations of antimicrobial resistance in clinically relevant bacterial pathogens.


Subject(s)
Acinetobacter baumannii/genetics , Drug Resistance, Multiple, Bacterial/genetics , Escherichia coli/genetics , Klebsiella pneumoniae/genetics , Oligonucleotide Array Sequence Analysis/methods , Acinetobacter Infections/drug therapy , Acinetobacter Infections/epidemiology , Acinetobacter Infections/microbiology , Acinetobacter baumannii/drug effects , Acinetobacter baumannii/enzymology , Acinetobacter baumannii/isolation & purification , Anti-Bacterial Agents/pharmacology , DNA, Bacterial/analysis , DNA, Bacterial/genetics , Egypt/epidemiology , Escherichia coli/drug effects , Escherichia coli/enzymology , Escherichia coli/isolation & purification , Escherichia coli Infections/drug therapy , Escherichia coli Infections/epidemiology , Escherichia coli Infections/microbiology , Humans , Klebsiella Infections/drug therapy , Klebsiella Infections/epidemiology , Klebsiella Infections/microbiology , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/enzymology , Klebsiella pneumoniae/isolation & purification , Polymerase Chain Reaction , Sequence Analysis, DNA , beta-Lactamases/genetics , beta-Lactamases/metabolism
7.
J Infect Dev Ctries ; 5(3): 169-75, 2011 Mar 21.
Article in English | MEDLINE | ID: mdl-21444985

ABSTRACT

INTRODUCTION: Typhoid fever is endemic in many parts of the world and represents a major cause of acute febrile illness (AFI). Rapid and accurate laboratory methods for diagnosis of this disease are needed for both patient care and surveillance situations. METHODOLOGY: Serum samples were collected from AFI patients and used to evaluate the performance of a newly developed ELISA assay that uses a mixture of somatic and flagellar antigens to detect the total antibody response against Salmonella enterica subspecies enterica serovar Typhi (S. Typhi) infection. The levels of Ig isotype response (IgG, IgM and IgA) were also evaluated, and results were compared to those of TUBEX-TF and Typhidot commercial kits.  RESULTS: Of 234 culture-confirmed typhoid patients, the total Ig ELISA diagnosed 93% compared to 71% using Widal test. This sensitivity level (93%) is higher than that observed for the individual Ig ELISAs (IgG 75%; IgM 79%; IgA 57%) and the commercial tests TUBEX-TF (75%), Typhidot IgM (63%) and Typhidot IgG (28%). An agreement of 78% was achieved between the total Ig ELISA and Widal test. The average specificity of the ELISA was 96%. Using ELISA, up to 200 samples can be tested per run with cost per test at US$0.20. CONCLUSIONS: The developed ELISA shows superior sensitivity and specificity, when compared to Widal, TUBEX-TF and Typhidot assays, is more cost effective and allows higher throughput. This method is highly recommended for active surveillance studies or outbreak investigations of typhoid fever.


Subject(s)
Clinical Laboratory Techniques/methods , Typhoid Fever/diagnosis , Adult , Antibodies, Bacterial/blood , Antigens, Bacterial , Enzyme-Linked Immunosorbent Assay/methods , Humans , Immunoglobulin A/blood , Immunoglobulin G/blood , Immunoglobulin M/blood , Salmonella typhi/immunology , Sensitivity and Specificity
8.
Trends Parasitol ; 25(11): 525-33, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19747878

ABSTRACT

For more than 25 years, the ISI assay and ILSDA have been used to study the development of the malaria parasite in the liver, to discover and characterize sporozoite and liver-stage antigens, to support the development of malaria vaccine candidates, and to search for immunological correlates of protection in animals and in humans. Although both assays have been limited by low sporozoite invasion rates, significant biological variability, and the subjective nature of manually counting hepatocytes containing parasites as the read-out, they have nevertheless been useful tools for exploring parasite biology. This review describes the origin, application and current status of these assays, critically discusses the need for improvements, and explores the roles of these assays in supporting the development of an effective vaccine against Plasmodium falciparum malaria.


Subject(s)
Immunoassay/methods , Liver/parasitology , Malaria, Falciparum/diagnosis , Plasmodium falciparum/isolation & purification , Humans , Malaria, Falciparum/parasitology , Sporozoites/growth & development
9.
Appl Environ Microbiol ; 71(10): 5858-64, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16204497

ABSTRACT

The nitrogen-fixing, symbiotic bacterium Sinorhizobium meliloti reduces molecular dinitrogen to ammonia in a specific symbiotic context, supporting the nitrogen requirements of various forage legumes, including alfalfa. Determining the DNA sequence of the S. meliloti genome was an important step in plant-microbe interaction research, adding to the considerable information already available about this bacterium by suggesting possible functions for many of the >6,200 annotated open reading frames (ORFs). However, the predictive power of bioinformatic analysis is limited, and putting the role of these genes into a biological context will require more definitive functional approaches. We present here a strategy for genetic analysis of S. meliloti on a genomic scale and report the successful implementation of the first step of this strategy by constructing a set of plasmids representing 100% of the 6,317 annotated ORFs cloned into a mobilizable plasmid by using efficient PCR and recombination protocols. By using integrase recombination to insert these ORFs into other plasmids in vitro or in vivo (B. L. House et al., Appl. Environ. Microbiol. 70:2806-2815, 2004), this ORFeome can be used to generate various specialized genetic materials for functional analysis of S. meliloti, such as operon fusions, mutants, and protein expression plasmids. The strategy can be generalized to many other genome projects, and the S. meliloti clones should be useful for investigators wanting an accessible source of cloned genes encoding specific enzymes.


Subject(s)
Cloning, Molecular/methods , Computational Biology/methods , Genome, Bacterial , Open Reading Frames/genetics , Recombination, Genetic , Sinorhizobium meliloti/genetics , Bacterial Proteins/genetics , DNA Primers , Databases, Genetic , Genetic Techniques , Genomics , Plasmids , Polymerase Chain Reaction
10.
Appl Environ Microbiol ; 70(5): 2806-15, 2004 May.
Article in English | MEDLINE | ID: mdl-15128536

ABSTRACT

The availability of bacterial genome sequences has created a need for improved methods for sequence-based functional analysis to facilitate moving from annotated DNA sequence to genetic materials for analyzing the roles that postulated genes play in bacterial phenotypes. A powerful cloning method that uses lambda integrase recombination to clone and manipulate DNA sequences has been adapted for use with the gram-negative alpha-proteobacterium Sinorhizobium meliloti in two ways that increase the utility of the system. Adding plasmid oriT sequences to a set of vehicles allows the plasmids to be transferred to S. meliloti by conjugation and also allows cloned genes to be recombined from one plasmid to another in vivo by a pentaparental mating protocol, saving considerable time and expense. In addition, vehicles that contain yeast Flp recombinase target recombination sequences allow the construction of deletion mutations where the end points of the deletions are located at the ends of the cloned genes. Several deletions were constructed in a cluster of 60 genes on the symbiotic plasmid (pSymA) of S. meliloti, predicted to code for a denitrification pathway. The mutations do not affect the ability of the bacteria to form nitrogen-fixing nodules on Medicago sativa (alfalfa) roots.


Subject(s)
Recombination, Genetic , Sinorhizobium meliloti/genetics , Acetylene/metabolism , Base Sequence , Conjugation, Genetic , DNA Nucleotidyltransferases/genetics , DNA Nucleotidyltransferases/metabolism , Escherichia coli/genetics , Gene Deletion , Medicago sativa/microbiology , Molecular Sequence Data , Nitrogen Fixation , Oxidation-Reduction , Plasmids/genetics
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