A Rapid Method for Quantifying Viable Mycobacterium avium subsp. paratuberculosis in Cellular Infection Assays

ABSTRACTDetermining the viability of bacteria is a key outcome ofin vitrocellular infection assays. Currently, this is done by culture, which is problematic for fastidious slow-growing bacteria such asMycobacterium aviumsubsp.paratuberculosis, where it can take up to 4 months to confirm growth. This study aimed to identify an assay that can rapidly quantify the number of viableM. aviumsubsp.paratuberculosiscells in a cellular sample. Three commercially available bacterial viability assays along with a modified liquid culture method coupled with high-throughput quantitative PCR growth detection were assessed. Criteria for assessment included the ability of each assay to differentiate live and deadM. aviumsubsp.paratuberculosisorganisms and their accuracy at low bacterial concentrations. Using the culture-based method,M. aviumsubsp.paratuberculosisgrowth was reliably detected and quantified within 2 weeks. There was a strong linear association between the 2-week growth rate and the initial inoculum concentration. The number of viableM. aviumsubsp.paratuberculosiscells in an unknown sample was quantified based on the growth rate, by using growth standards. In contrast, none of the commercially available viability assays were suitable for use with samples fromin vitrocellular infection assays.IMPORTANCERapid quantification of the viability ofMycobacterium aviumsubsp.paratuberculosisin samples fromin vitrocellular infection assays is important, as it allows these assays to be carried out on a large scale.In vitrocellular infection assays can function as a preliminary screening tool, for vaccine development or antimicrobial screening, and also to extend findings derived from experimental animal trials. Currently, by using culture, it takes up to 4 months to obtain quantifiable results regardingM. aviumsubsp.paratuberculosisviability after anin vitroinfection assay; however, with the quantitative PCR and liquid culture method developed, reliable results can be obtained at 2 weeks. This method will be important for vaccine and antimicrobial screening work, as it will allow a greater number of candidates to be screened in the same amount of time, which will increase the likelihood that a favorable candidate will be found to be subjected to further testing.

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Enhancement of Campylobacter hepaticus culturing to facilitate downstream applications

Scientific Reports ◽

10.1038/s41598-021-00277-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Canh Phung ◽

Timothy B. Wilson ◽

José A. Quinteros ◽

Peter C. Scott ◽

Robert J. Moore ◽

...

Keyword(s):

Liquid Culture ◽

Vaccine Development ◽

Genomic Analysis ◽

Bacterial Biomass ◽

In Silico Analysis ◽

Culture Method ◽

Efficient Production ◽

Culture Density ◽

Brucella Broth

AbstractCampylobacter hepaticus causes Spotty Liver Disease (SLD) in chickens. C. hepaticus is fastidious and slow-growing, presenting difficulties when growing this bacterium for the preparation of bacterin vaccines and experimental disease challenge trials. This study applied genomic analysis and in vitro experiments to develop an enhanced C. hepaticus liquid culture method. In silico analysis of the anabolic pathways encoded by C. hepaticus revealed that the bacterium is unable to biosynthesise l-cysteine, l-lysine and l-arginine. It was found that l-cysteine added to Brucella broth, significantly enhanced the growth of C. hepaticus, but l-lysine or l-arginine addition did not enhance growth. Brucella broth supplemented with l-cysteine (0.4 mM), l-glutamine (4 mM), and sodium pyruvate (10 mM) gave high-density growth of C. hepaticus and resulted in an almost tenfold increase in culture density compared to the growth in Brucella broth alone (log10 = 9.3 vs 8.4 CFU/mL). The type of culture flask used also significantly affected C. hepaticus culture density. An SLD challenge trial demonstrated that C. hepaticus grown in the enhanced culture conditions retained full virulence. The enhanced liquid culture method developed in this study enables the efficient production of bacterial biomass and therefore facilitates further studies of SLD biology and vaccine development.

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Isolation of Mycobacterium avium Subsp. Paratuberculosis in the Feces and Tissue of Small Ruminants Using a Non-Automated Liquid Culture Method

Animals ◽

10.3390/ani10010020 ◽

2019 ◽

Vol 10 (1) ◽

pp. 20

Author(s):

Luigi De Grossi ◽

Davide Santori ◽

Antonino Barone ◽

Silvia Abbruzzese ◽

Matteo Ricchi ◽

...

Keyword(s):

Mycobacterium Avium ◽

Liquid Culture ◽

Culture Media ◽

Small Ruminants ◽

Culture Method ◽

Mycobacterium Avium Subsp ◽

Type I ◽

Culture Methods ◽

Specific Pcr ◽

Mycobacterium Avium Subsp Paratuberculosis

Paratuberculosis is a chronic disease of ruminants caused by Mycobacterium avium subsp. Paratuberculosis (MAP). Since isolation of MAP type I (S) is rarely reported in Italy, our research was aimed at isolating, by an inexpensive liquid culture manual method, this type of MAP isolates. At first, we used an ELISA to point out to serologically positive samples from five flocks. Secondly, we used a fecal direct IS900-qPCR on the ELISA positive samples, in order to detect shedder animals. Feces from IS900-qPCR positive samples were inoculated in solid and liquid culture media. IS900-qPCR was further used to test the growth of MAP isolates in liquid medium, which were further confirmed by f57-qPCR and submitted to typing by specific PCR in order to identify the MAP type. Twenty-eight samples (24 fecal and four tissutal samples) were processed by culture methods, resulting in the isolation of six type I MAP field isolates. Notably, no isolates were recovered by solid media, underlining the utility of this liquid method. Few data about this type of MAP are currently available in Italy, and further analyses should be carried out in order to study the origin and epidemiology of type I strains circulating in Italy.

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Biological Impact of a Large-Scale Genomic Inversion That Grossly Disrupts the Relative Positions of the Origin and Terminus Loci of theStreptococcus pyogenesChromosome

Journal of Bacteriology ◽

10.1128/jb.00090-19 ◽

2019 ◽

Vol 201 (17) ◽

Cited By ~ 1

Author(s):

Dragutin J. Savic ◽

Scott V. Nguyen ◽

Kimberly McCullor ◽

W. Michael McShan

Keyword(s):

Dna Sequences ◽

Parental Strain ◽

Large Scale ◽

Galleria Mellonella ◽

Acute Infection ◽

Relative Length ◽

Published Data ◽

Rich Medium ◽

Content Type

ABSTRACTA large-scale genomic inversion encompassing 0.79 Mb of the 1.816-Mb-longStreptococcus pyogenesserotype M49 strain NZ131 chromosome spontaneously occurs in a minor subpopulation of cells, and in this report genetic selection was used to obtain a stable lineage with this chromosomal rearrangement. This inversion, which drastically displaces theorisite relative to the terminus, changes the relative length of the replication arms so that one replichore is approximately 0.41 Mb while the other is about 1.40 Mb in length. Genomic reversion to the original chromosome constellation is not observed in PCR-monitored analyses after 180 generations of growth in rich medium. Compared to the parental strain, the inversion surprisingly demonstrates a nearly identical growth pattern in the first phase of the exponential phase, but differences do occur when resources in the medium become limited. When cultured separately in rich medium during prolonged stationary phase or in an experimental acute infection animal model (Galleria mellonella), the parental strain and the invertant have equivalent survival rates. However, when they are coincubated together, bothin vitroandin vivo, the survival of the invertant declines relative to the level for the parental strain. The accompanying aspect of the study suggests that inversions taking place nearoriCalways happen to secure the linkage oforiCto DNA sequences responsible for chromosome partition. The biological relevance of large-scale inversions is also discussed.IMPORTANCEBased on our previous work, we created to our knowledge the largest asymmetric inversion, covering 43.5% of theS. pyogenesgenome. In spite of a drastic replacement of origin of replication and the unbalanced size of replichores (1.4 Mb versus 0.41 Mb), the invertant, when not challenged with its progenitor, showed impressive vitality for growthin vitroand in pathogenesis assays. The mutant supports the existing idea that slightly deleterious mutations can provide the setting for secondary adaptive changes. Furthermore, comparative analysis of the mutant with previously published data strongly indicates that even large genomic rearrangements survive provided that the integrity of theoriCand the chromosome partition cluster is preserved.

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Effective Treatment of Mycobacterium avium subsp. hominissuis and Mycobacterium abscessus Species Infections in Macrophages, Biofilm, and Mice by Using Liposomal Ciprofloxacin

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00440-18 ◽

2018 ◽

Vol 62 (10) ◽

Cited By ~ 9

Author(s):

James D. Blanchard ◽

Valerie Elias ◽

David Cipolla ◽

Igor Gonda ◽

Luiz E. Bermudez

Keyword(s):

Effective Treatment ◽

Mouse Models ◽

Mycobacterium Avium ◽

Nontuberculous Mycobacteria ◽

Mycobacterium Abscessus ◽

Content Type ◽

Number Of Individuals ◽

Intranasal Instillation

ABSTRACT Nontuberculous mycobacteria (NTM) affect an increasing number of individuals worldwide. Infection with these organisms is more common in patients with chronic lung conditions, and treatment is challenging. Quinolones, such as ciprofloxacin, have been used to treat patients, but the results have not been encouraging. In this report, we evaluate novel formulations of liposome-encapsulated ciprofloxacin (liposomal ciprofloxacin) in vitro and in vivo. Its efficacy against Mycobacterium avium and Mycobacterium abscessus was examined in macrophages, in biofilms, and in vivo using intranasal instillation mouse models. Liposomal ciprofloxacin was significantly more active than free ciprofloxacin against both pathogens in macrophages and biofilms. When evaluated in vivo, treatment with the liposomal ciprofloxacin formulations was associated with significant decreases in the bacterial loads in the lungs of animals infected with M. avium and M. abscessus. In summary, topical delivery of liposomal ciprofloxacin in the lung at concentrations greater than those achieved in the serum can be effective in the treatment of NTM, and further evaluation is warranted.

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High speed large scale automated isolation of SARS-CoV-2 from clinical samples using miniaturized co-culture coupled with high content screening

10.1101/2020.05.14.097295 ◽

2020 ◽

Cited By ~ 2

Author(s):

Rania Francis ◽

Marion Le Bideau ◽

Priscilla Jardot ◽

Clio Grimaldier ◽

Didier Raoult ◽

...

Keyword(s):

High Speed ◽

Large Scale ◽

Vaccine Development ◽

Work Load ◽

Drug Susceptibility Testing ◽

High Content Screening ◽

Clinical Samples ◽

Rt Pcr ◽

Positive Elements

AbstractSARS-CoV-2, a novel coronavirus infecting humans, is responsible for the current COVID-19 global pandemic. If several strains could be isolated worldwide, especially for in-vitro drug susceptibility testing and vaccine development, few laboratories routinely isolate SARS-CoV-2. This is due to the fact that the current co-culture strategy is highly time consuming and requires working in a biosafety level 3 laboratory. In this work, we present a new strategy based on high content screening automated microscopy (HCS) allowing large scale isolation of SARS-CoV-2 from clinical samples in 1 week. A randomized panel of 104 samples, including 72 tested positive by RT-PCR and 32 tested negative, were processed with our HCS procedure and were compared to the classical isolation procedure. Isolation rate was 43 % with both strategies on RT-PCR positive samples, and was correlated with the initial RNA viral load in the samples, where we obtained a positivity threshold of 27 Ct. Co-culture delays were shorter with HCS strategy, where 80 % of the positive samples were recovered by the third day of co-culture, as compared to only 25 % with the classic strategy. Moreover, only the HCS strategy allowed us to recover all the positive elements after 1 week of co-culture. This system allows rapid and automated screening of clinical samples with minimal operator work load, thus reducing the risks of contamination.

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In Vitro Activity of Rifamycin Derivatives against Nontuberculous Mycobacteria, Including Macrolide- and Amikacin-Resistant Clinical Isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02611-20 ◽

2021 ◽

Vol 65 (5) ◽

Author(s):

Dae Hun Kim ◽

Su-Young Kim ◽

Hee Jae Huh ◽

Nam Yong Lee ◽

Won-Jung Koh ◽

...

Keyword(s):

Mycobacterium Avium ◽

Nontuberculous Mycobacteria ◽

Therapeutic Option ◽

Vitro Activity ◽

Clinical Isolates ◽

Drug Resistant ◽

In Vitro Activity ◽

Content Type ◽

Resistant Disease

ABSTRACT We evaluated the in vitro activity of rifamycin derivatives, including rifampin, rifapentine, rifaximin, and rifabutin, against clinical nontuberculous mycobacteria (NTM) isolates. Of the rifamycin derivatives, rifabutin showed the lowest MICs against all NTM species, including Mycobacterium avium complex, M. abscessus, and M. kansasii. Rifabutin also had effective in vitro activity against macrolide- and aminoglycoside-resistant NTM isolates. Rifabutin could be worth considering as a therapeutic option for NTM disease, particularly drug-resistant disease.

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The Combinations Chitosan-Pam3CSK4 and Chitosan-Monophosphoryl Lipid A: Promising Immune-Enhancing Adjuvants for Anticaries Vaccine PAc

Infection and Immunity ◽

10.1128/iai.00651-19 ◽

2019 ◽

Vol 87 (12) ◽

Author(s):

Yongli Bi ◽

Qingan Xu ◽

Lingkai Su ◽

Jiantao Xu ◽

Zhongfang Liu ◽

...

Keyword(s):

Lipid A ◽

Vaccine Development ◽

Protection Effect ◽

Content Type ◽

Monophosphoryl Lipid A ◽

Monophosphoryl Lipid ◽

Tooth Surfaces ◽

Antibody Titers

ABSTRACT We previously demonstrated that recombinant protein PAc could be administered as an anticaries vaccine. However, the relatively weak immunogenicity of PAc limits its application. In the present study, we investigated the effect of two adjuvant combinations of chitosan plus Pam3CSK4 (chitosan-Pam3CSK4) and of chitosan plus monophosphoryl lipid A (chitosan-MPL) in the immune responses to the PAc protein in vivo and in vitro. PAc-chitosan-Pam3CSK4 or PAc-chitosan-MPL promoted significantly higher PAc-specific antibody titers in serum and saliva, inhibited Streptococcus mutans colonization onto the tooth surfaces, and endowed better protection effect with significantly less caries activities than PAc alone. Chitosan-Pam3CSK4 and chitosan-MPL showed no statistically significant differences. In conclusion, our study demonstrated that the chitosan-Pam3CSK4 and chitosan-MPL combinations are promising for anticaries vaccine development.

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Decoration of Outer Membrane Vesicles with Multiple Antigens by Using an Autotransporter Approach

Applied and Environmental Microbiology ◽

10.1128/aem.01941-14 ◽

2014 ◽

Vol 80 (18) ◽

pp. 5854-5865 ◽

Cited By ~ 41

Author(s):

Maria H. Daleke-Schermerhorn ◽

Tristan Felix ◽

Zora Soprova ◽

Corinne M. ten Hagen-Jongman ◽

David Vikström ◽

...

Keyword(s):

Immune Responses ◽

Outer Membrane ◽

Vaccine Development ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Heterologous Proteins ◽

Content Type ◽

Serovar Typhimurium ◽

Heterologous Antigens

ABSTRACTOuter membrane vesicles (OMVs) are spherical nanoparticles that naturally shed from Gram-negative bacteria. They are rich in immunostimulatory proteins and lipopolysaccharide but do not replicate, which increases their safety profile and renders them attractive vaccine vectors. By packaging foreign polypeptides in OMVs, specific immune responses can be raised toward heterologous antigens in the context of an intrinsic adjuvant. Antigens exposed at the vesicle surface have been suggested to elicit protection superior to that from antigens concealed inside OMVs, but hitherto robust methods for targeting heterologous proteins to the OMV surface have been lacking. We have exploited our previously developed hemoglobin protease (Hbp) autotransporter platform for display of heterologous polypeptides at the OMV surface. One, two, or three of theMycobacterium tuberculosisantigens ESAT6, Ag85B, and Rv2660c were targeted to the surface ofEscherichia coliOMVs upon fusion to Hbp. Furthermore, a hypervesiculating ΔtolRΔtolAderivative of attenuatedSalmonella entericaserovar Typhimurium SL3261 was generated, enabling efficient release and purification of OMVs decorated with multiple heterologous antigens, exemplified by theM. tuberculosisantigens and epitopes fromChlamydia trachomatismajor outer membrane protein (MOMP). Also, we showed that delivery ofSalmonellaOMVs displaying Ag85B to antigen-presenting cellsin vitroresults in processing and presentation of an epitope that is functionally recognized by Ag85B-specific T cell hybridomas. In conclusion, the Hbp platform mediates efficient display of (multiple) heterologous antigens, individually or combined within one molecule, at the surface of OMVs. Detection of antigen-specific immune responses upon vesicle-mediated delivery demonstrated the potential of our system for vaccine development.

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Cooperative Role of Antibodies against Heat-Labile Toxin and the EtpA Adhesin in Preventing Toxin Delivery and Intestinal Colonization by Enterotoxigenic Escherichia coli

Clinical and Vaccine Immunology ◽

10.1128/cvi.00351-12 ◽

2012 ◽

Vol 19 (10) ◽

pp. 1603-1608 ◽

Cited By ~ 20

Author(s):

Koushik Roy ◽

David J. Hamilton ◽

James M. Fleckenstein

Keyword(s):

Escherichia Coli ◽

Diarrheal Disease ◽

Vaccine Development ◽

Enterotoxigenic Escherichia Coli ◽

Intestinal Colonization ◽

Content Type ◽

Heat Labile

ABSTRACTEnterotoxigenicEscherichia coli(ETEC) is an important cause of diarrheal disease in developing countries, where it is responsible for hundreds of thousands of deaths each year. Vaccine development for ETEC has been hindered by the heterogeneity of known molecular targets and the lack of broad-based sustained protection afforded by existing vaccine strategies. In an effort to explore the potential role of novel antigens in ETEC vaccines, we examined the ability of antibodies directed against the ETEC heat-labile toxin (LT) and the recently described EtpA adhesin to prevent intestinal colonizationin vivoand toxin delivery to epithelial cellsin vitro. We demonstrate that EtpA is required for the optimal delivery of LT and that antibodies against this adhesin play at least an additive role in preventing delivery of LT to target intestinal cells when combined with antibodies against either the A or B subunits of the toxin. Moreover, vaccination with a combination of LT and EtpA significantly impaired intestinal colonization. Together, these results suggest that the incorporation of recently identified molecules such as EtpA could be used to enhance current approaches to ETEC vaccine development.

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Development, Interlaboratory Evaluations, and Application of a Simple, High-ThroughputShigellaSerum Bactericidal Assay

mSphere ◽

10.1128/msphere.00146-18 ◽

2018 ◽

Vol 3 (3) ◽

Cited By ~ 6

Author(s):

Moon H. Nahm ◽

Jigui Yu ◽

Hailey P. Weerts ◽

Heather Wenzel ◽

Chitradevi S. Tamilselvi ◽

...

Keyword(s):

Vaccine Development ◽

Shigella Flexneri ◽

Binding Activity ◽

Serum Samples ◽

Bacterial Killing ◽

Content Type ◽

Bactericidal Assay ◽

Serum Bactericidal Assay ◽

Reference Serum

ABSTRACTShigellais an important cause of diarrhea worldwide, with serotypesShigella flexneri2a,S. flexneri3a, andShigella sonneidemonstrating epidemiological prevalence. Many development efforts are focused onShigellalipopolysaccharide (LPS)-based vaccines, as O antigen-specific conjugate vaccines are immunogenic and efficacious. Immunization withShigellavaccines containing LPS can elicit antibodies capable of killingShigellain a serotype-specific manner. Thus, to facilitateShigellavaccine development, we have developed a serum bactericidal assay (SBA) specific for threeShigellaserotypes that measures killing of target bacteria at multiple serum dilutions and in the presence of exogenous complement. The SBA has a high analytical throughput and uses simple technologies and readily available reagents. The SBA was characterized with human sera with bactericidal antibodies againstS. flexneri2a,S. flexneri3a, andS. sonnei. Purified LPS of a homologous serotype, but not a heterologous serotype, inhibited bacterial killing. Assessment of precision found median intra-assay precision to be 13.3% and median interassay precision to be 19 to 30% for the three serotypes. The SBA is linear, with slight deviations for samples with low (~40) killing indices. The SBA was sensitive enough to allow about 100-fold predilution of serum samples. Repeat assays yielded results with less than 2-fold deviations, indicating the robustness of the assay. Assay results from four different laboratories were highly comparable when normalized with a reference serum. TheShigellaSBA, combined with a reference serum, should facilitate the development ofShigellavaccines across the field.IMPORTANCEShigellais an important cause of diarrhea worldwide, and efforts are ongoing to produce a safe and effectiveShigellavaccine. Although a clear immune correlate of protection has not been established, antibodies with bactericidal capacity may provide one means of protecting against shigellosis. Thus, it is important to measure the functional capacity of antibodies, as opposed to only binding activity. This article describes a simple, robust, and high-throughput serum bactericidal assay capable of measuringShigella-specific functional antibodiesin vitro. We show for the first time that this assay was successfully performed by multiple laboratories and generated highly comparable results, particularly when SBA titers were normalized using a reference standard. The serum bactericidal assay, along with a reference serum, should greatly facilitateShigellavaccine development.

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