Low‐cost, high‐throughput, automated counting of bacterial colonies

Clarke, Matthew L.; Burton, Robert L.; Hill, Nayo M.; Litorja, Maritoni; Nahm, Moon H.; Hwang, Jeeseong

doi:10.1002/cyto.a.20864

Cited by 97 publications

(107 citation statements)

References 33 publications

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“…All unvaccinated human sera were 2-fold prediluted, all postvaccination human sera were 20-fold prediluted, fs15b was 225-fold prediluted, and Hyp15GB5 was 20-fold prediluted. Colonies were counted using NICE software (63), and OIs were calculated using Opsotiter (A1.0 NICE) (62).…”

Section: Methodsmentioning

confidence: 99%

The Pneumococcal Serotype 15C Capsule Is Partially O-Acetylated and Allows for Limited Evasion of 23-Valent Pneumococcal Polysaccharide Vaccine-Elicited Anti-Serotype 15B Antibodies

Spencer

Shenoy

Orihuela

et al. 2017

Clin Vaccine Immunol

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As a species, Streptococcus pneumoniae (the pneumococcus) utilizes a diverse array of capsular polysaccharides to evade the host. In contrast to large variations in sugar composition and linkage formation, O-acetylation is a subtle capsular modification that nonetheless has a large impact on capsular shielding and recognition of the capsule by vaccine-elicited antibodies. Serotype 15B, which is included in the 23-valent pneumococcal polysaccharide vaccine (PPV23), carries the putative O-acetyltransferase gene wciZ. The coding sequence of wciZ contains eight consecutive TA repeats [(TA) 8 ]. Replication slippage is thought to result in the addition or loss of TA repeats, subsequently causing frameshift and truncation of WciZ to yield a nonacetylated serotype, 15C. Using sensitive serological tools, we show that serotype 15C isolates whose wciZ contains seven or nine TA repeats retain partial O-acetylation, while serotype 15C isolates whose wciZ contains six TA repeats have barely detectable O-acetylation. We confirmed by inhibition enzyme-linked immunosorbent assay that (TA) 7 serotype 15C is ϳ0.1% as acetylated as serotype 15B, while serotype 15X is nonacetylated. To eliminate the impact of genetic background, we created isogenic serotype 15B, (TA) 7 serotype 15C, and 15BΔwciZ (15X) strains and found that reduction or absence of WciZ-mediated O-acetylation did not affect capsular shielding from phagocytes, biofilm formation, adhesion to nasopharyngeal cells, desiccation tolerance, or murine colonization. Sera from PPV23-immunized persons opsonized serotype 15B significantly but only slightly better than serotypes 15C and 15X; thus, PPV23 may not result in expansion of serotype 15C.KEYWORDS O-acetyltransferase, O-acetylation, capsule diversity, pneumococcal vaccine, capsular polysaccharide, serotyping, replication slippage S treptococcus pneumoniae (the pneumococcus), a Gram-positive human pathogen, mediates pneumonia and invasive diseases such as septicemia and meningitis and colonizes 6 to 76% of the world's pediatric population (1-3). Pneumococcal diseases are the leading cause of death in children under 5 years old (4), and pneumococcal capsular polysaccharide is the organism's most significant virulence factor as it shields the pneumococcus from various chemical and immune assaults (5). As a result, nonencapsulated pneumococci rarely cause pneumonia or invasive pneumococcal disease (6-8). Pneumococcal capsular types differ in their shielding abilities; therefore, some capsule types are more virulent than others (9, 10). Our previous studies have indicated that small chemical modifications of the capsular repeat unit result in differential shielding of closely related serotypes (11,12).An important capsular modification is O-acetylation: an uncharged, but polar, acetyl

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Section: Methodsmentioning

confidence: 99%

The Pneumococcal Serotype 15C Capsule Is Partially O-Acetylated and Allows for Limited Evasion of 23-Valent Pneumococcal Polysaccharide Vaccine-Elicited Anti-Serotype 15B Antibodies

Spencer

Shenoy

Orihuela

et al. 2017

Clin Vaccine Immunol

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“…The agar plate was then overlaid with agar containing 0.1% NaN 3 and 100 g/ml of triphenyltetrazolium chloride (TTC), which is reduced by the growing bacteria to a red compound resulting in colored colonies. The petri dish was photographed with a digital camera, and the number of colonies was determined with a computer program called NICE (NIST's integrated colony enumerator) (20).…”

Section: Methodsmentioning

confidence: 99%

Monoclonal Antibodies to Shigella Lipopolysaccharide Are Useful for Vaccine Production

Lin

Smith

Benjamin

et al. 2016

Clin Vaccine Immunol

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There is a significant need for an effective multivalent Shigella vaccine that targets the most prevalent serotypes. Most Shigella vaccines under development utilize serotype-specific lipopolysaccharides (LPSs) as a major component based on protection and epidemiological data. As vaccine formulations advance from monovalent to multivalent, assays and reagents need to be developed to accurately and reproducibly quantitate the amount of LPSs from multiple serotypes in the final product. To facilitate this effort, we produced 36 hybridomas that secrete monoclonal antibodies (MAbs) against the O antigen on the LPS from Shigella flexneri 2a, Shigella flexneri 3a, and Shigella sonnei. We used six of these monoclonal antibodies for an inhibition enzymelinked immunosorbent assay (iELISA), measuring LPSs with high sensitivity and specificity. It was also demonstrated that the Shigella serotype-specific MAbs were useful for bacterial surface staining detected by flow cytometry. These MAbs are also useful for standardizing the serum bactericidal assay (SBA) for Shigella. Functional assays, such as the in vitro bactericidal assay, are necessary for vaccine evaluation and may serve as immunological correlates of immunity. An S. flexneri 2a-specific monoclonal antibody killed S. flexneri 2b isolates, suggesting that S. flexneri 2a LPS may induce cross-protection against S. flexneri 2b. Overall, the Shigella LPS-specific MAbs described have potential utility to the vaccine development community for assessing multivalent vaccine composition and as a reliable control for multiple immunoassays used to assess vaccine potency.

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“…Up to now, the majority of HW/SW imaging solutions related to bacteria colony growth have been focused on providing automated colony counting features, especially for application fields relying on exact (quantitative) and fast bacterial load estimation, such as food and beverage safety [2], [3], [4] or various environmental control and specific clinical usages [5], [6], [7]. Clinical bacteriology usually requires qualitative bacterial load estimation even in cases where massively confluent colonies growth occurs and traditional bacterial enumeration systems would fail.…”

Section: Related Workmentioning

confidence: 99%

Multistage classification for bacterial colonies recognition on solid agar images

Ferrari

Signoroni

2014

2014 IEEE International Conference on Imaging Systems and Techniques (IST) Proceedings

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Abstract-The advent of laboratory automation in clinical microbiology is entailing a revolution in the way most common bacteriological clinical exams are accomplished. As an essential part of these systems, digital recording and processing of cultured bacteria images is expected to improve plate reading, with a key role of image analysis tools in guaranteeing cost-effectiveness, accuracy, flexibility and reliability of the clinical tasks. In this work, we propose an image analysis system capable to address the complex problem of different bacteria species identification on cultured agar plates. Our solution is based on a modular segmentation/classification pipeline where a chain of supervised classification stages provides solutions to a series of nested task issues, from foreground separation toward isolated colony detection and classification. Performance assessment, based on an experimental dataset obtained in standardized laboratory conditions, clearly demonstrates the feasibility and the potentiality of the proposed solution and favorably opens to generalizations as well as to clinical validation studies.

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Low‐cost, high‐throughput, automated counting of bacterial colonies

Cited by 97 publications

References 33 publications

The Pneumococcal Serotype 15C Capsule Is Partially O-Acetylated and Allows for Limited Evasion of 23-Valent Pneumococcal Polysaccharide Vaccine-Elicited Anti-Serotype 15B Antibodies

The Pneumococcal Serotype 15C Capsule Is Partially O-Acetylated and Allows for Limited Evasion of 23-Valent Pneumococcal Polysaccharide Vaccine-Elicited Anti-Serotype 15B Antibodies

Monoclonal Antibodies to Shigella Lipopolysaccharide Are Useful for Vaccine Production

Multistage classification for bacterial colonies recognition on solid agar images

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