Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal epidemiology, capsular serotyping can provide useful information for vaccine efficacy and impact studies. The Quellung reaction is the gold standard method for pneumococcal capsular serotyping. The method involves testing a pneumococcal cell suspension with pooled and specific antisera directed against the capsular polysaccharide. The antigen-antibody reactions are observed microscopically. The protocol has three main steps: 1) preparation of a bacterial cell suspension, 2) mixing of cells and antisera on a glass slide, and 3) reading the Quellung reaction using a microscope. The Quellung reaction is reasonably simple to perform and can be applied wherever a suitable microscope and antisera are available.     

Elucidation of structural and antigenic properties of pneumococcal serotype 11A, 11B, 11C, and 11F polysaccharide capsules

Despite the emerging impact of serogroup 11 serotypes in Streptococcus pneumoniae epidemiology, the structures of serogroup 11 capsule types have not been fully elucidated, particularly the locations of O-acetyl substitutions. Here, we report the complete structures of the serotype 11B, 11C, and 11F polysaccharides and a revision to the serotype 11A capsular polysaccharide using nuclear magnetic resonance (NMR). All structures shared a linear, tetrasaccharide backbone with a pendant phosphopolyalcohol. Three of four saccharides are conserved in all serotypes. The individual serotype capsules differed in the identity of one saccharide, the pendant phosphopolyalcohol, and the O-acetylation pattern. Though the assigned locations of O-acetate substitutions in this study differed from those of previous reports, our findings were corroborated with strong correlations to serology and genetics. We examined the binding of serotyping sera to serogroup 11 polysaccharides by using flow cytometry and an inhibition-type enzyme-linked immunosorbent assay (ELISA) and found that de-O-acetylation of capsular polysaccharides by mild hydrolysis decreases its immunoreactivity, supporting the crucial role of O-acetylation in the antigenicity of these polysaccharides. Due to strong correlations between polysaccharide structures and capsule biosynthesis genes, we were able to assign target substrates for the O-acetyltransferases encoded by wcwC, wcwR, wcwT, and wcjE. We identified antigenic determinants for serogroup 11 serotyping sera and highlight the idea that conventional serotyping methods are not capable of recognizing all putative variants of S. pneumoniae serogroup 11.     

Antigenic structure of the polysaccharide capsule of Streptococcus pneumoniae strains isolated in Leningrad 1978-1984

The serotyping of 826 S. pneumoniae strains, isolated in conditionally diagnostic concentrations from the bronchial contents of patients with acute and chronic inflammatory pulmonary diseases during 1978-1984 in Leningrad, was made. The study revealed the prevalence of serotypes and groups 6, 23, 9, 3, 19, 15 and the undulant character of fluctuations in their annual occurrence. The specific proportion of the prevailing serotypes of S. pneumoniae among the cultures isolated from patients with acute pneumonia and acute bronchitis (6 and 19) was found to differ from that among S. pneumoniae strains isolated from patients with chronic bronchitis; in the latter patients serotypes 3 and 9 occurred more frequently (P less than 0.01).     

Structure of aggregates of Tipula iridescent virus and polystyrene latex particles

Tipula iridescent virus aggregated with polystyrene latex particles 126 nm in diameter. There was a region of optimal proportion of the two particles. The aggregation proceeded faster and the amount of resultant aggregates was larger at the higher concentrations of the two particles, but the size of the individual aggregates was smaller. The aggregates consisted of clusters of the two particles with vacant spaces interspersed among them. A hypothetical model of the particle arrangements was presented. The aggregates were reversibly dissolved in 0.03% sodium dodecyl sulfate and 30% n-propanol and isopropanol. In the presence of lower concentrations of these solvents, the aggregation occurred at high temperatures but not at low temperatures. These results were interpreted as implicating hydrophobic interactions in the formation and stability of the aggregates.     

致肝脓肿高毒力肺炎克雷伯菌的表型及分子特征

目的 探究导致肝脓肿高毒力肺炎克雷伯菌的分子特征。方法 用VITEK-2细菌鉴定仪鉴定2014年1月至2016年1月丽水市中心医院肝脓肿患者脓肿穿刺液中分离的细菌。应用拉丝试验鉴定菌株的高黏性,用多位点序列分型(MLST)和血清型分型(K分型)对菌株进行分子分型,并用S1核酸酶脉冲场凝胶电泳(S1-PFGE)对菌株质粒谱进行分析。结果 57例肝脓肿患者接受肝脏脓肿穿刺引流并做脓液培养。44例患者的脓液培养到不同的致病菌,培养阳性率为77.2%。在培养到的44株病原菌中,其中2株为大肠埃希菌,产酸克雷伯菌、金黄色葡萄球菌各1株,而肺炎克雷伯菌为40株,占肝脓肿致病菌的90.9%。40株肺炎克雷伯菌中,拉丝阳性率为67.5%(27/40),K1为主要血清分型,占62.5%(25/40),其次为K2型,占17.5%(7/40)。ST23为主要ST分型,占47.5%(19/40),其次为ST86和ST65,各占7.5%(3/40)。同时发现一些未报道过的致肝脓肿肺炎克雷伯菌新ST分型,如ST218、ST1941、ST76、ST2159、ST660和ST485。40株致肝脓肿肺炎克雷伯菌中总共检测到12种质粒谱,包括带有一个质粒、多个质粒或不带质粒的谱型。其中带有一个近220 kb的质粒谱为主要谱型,共涉及19株菌,占47.5%,12株菌带有一个质粒,大小为140～250 kb。4株菌带有2个或3个质粒,5株菌不含有质粒。结论 拉丝试验和血清学分型不能鉴定所有的高毒力肺炎克雷伯菌;高毒力肺炎克雷伯菌很多为ST23型,但其进化整体上较为分散;高毒力肺炎克雷伯菌菌株可以不携带质粒。     

Validity of an enzyme-linked immunosorbent assay with serotype-specific monoclonal antibodies for serotyping human rotavirus in stool specimens

We recently developed a method for serotyping human rotavirus (HRV) by an enzyme-linked immunosorbent assay with HRV serotype-specific neutralizing monoclonal antibodies (ELISA serotyping). In the present study this method was compared with the fluorescent focus neutralization test with serotype-specific rabbit antisera (NT serotyping) in the sensitivity and specificity of the test. Direct serotyping of HRVs which were contained in stool specimens indicated that while only 37% of the samples were successfully serotyped in NT, 78% of the samples could be serotyped in ELISA. Regarding the samples whose serotype could be determined in the two tests, the assigned serotypes were identical in both tests. The results obtained indicated the utility of ELISA serotyping in clinical and epidemiologic studies of HRV infection.     

Population-Based Analysis of Invasive Nontypeable Pneumococci Reveals That Most Have Defective Capsule Synthesis Genes

Since nasopharyngeal carriage of pneumococcus precedes invasive pneumococcal disease, characteristics of carriage isolates could be incorrectly assumed to reflect those of invasive isolates. While most pneumococci express a capsular polysaccharide, nontypeable pneumococci are sometimes isolated. Carriage nontypeables tend to encode novel surface proteins in place of a capsular polysaccharide synthetic locus, the cps locus. In contrast, capsular polysaccharide is believed to be indispensable for invasive pneumococcal disease, and nontypeables from population-based invasive pneumococcal disease surveillance have not been extensively characterized. We received 14,328 invasive pneumococcal isolates through the Active Bacterial Core surveillance program during 2006–2009. Isolates that were nontypeable by Quellung serotyping were characterized by PCR serotyping, sequence analyses of the cps locus, and multilocus sequence typing. Eighty-eight isolates were Quellung-nontypeable (0.61%). Of these, 79 (89.8%) contained cps loci. Twenty-two nontypeables exhibited serotype 8 cps loci with defects, primarily within wchA. Six of the remaining nine isolates contained previously-described aliB homologs in place of cps loci. Multilocus sequence typing revealed that most nontypeables that lacked capsular biosynthetic genes were related to established non-encapsulated lineages. Thus, invasive pneumococcal disease caused by nontypeable pneumococcus remains rare in the United States, and while carriage nontypeables lacking cps loci are frequently isolated, such nontypeable are extremely rare in invasive pneumococcal disease. Most invasive nontypeable pneumococci possess defective cps locus genes, with an over-representation of defective serotype 8 cps variants.     

Improved methods for detection and serotyping of coagulase from Staphylococcus aureus

Improved methods for detection and serotyping of staphylocoagulase were concomitantly devised. We devised an improved method for detection of coagulase activity on agarose film in the same manner as single radial immunodiffusion. The amounts of reagents required for detection of coagulase on agarose film were successfully diminished by adding polyethylene glycol (PEG) to the original formula described by Boothby et al. Using microplates in another improved method for coagulase serotyping, the amount of reaction fluid required was considerably less compared with the conventional tube method. PEG was found to be also effective to increase the efficacy of coagulase serotyping. In the presence or absence of anti-coagulase antisera, culture supernatants of staphylococcal strain grown in brain heart infusion broth were incubated with the reaction fluid containing bovine fibrinogen, rabbit plasma, 6-amino caproic acid, polyethylene glycol 6,000. Coagulase activity was visualized as a turbid mass formed in the wells. Turbid mass formation due to coagulase activity was type-specifically inhibited in the presence of type-specific antisera. Detailed procedures of the methods are precisely described with some preliminary results obtained by the methods.     

Construction of otherwise isogenic serotype 6B, 7F, 14, and 19F capsular variants of Streptococcus pneumoniae strain TIGR4

The polysaccharide capsule is the primary virulence factor in Streptococcus pneumoniae. There are at least 90 serotypes of S. pneumoniae, identified based on the immunogenicity of different capsular sugars. The aim of this study was to construct pneumococcal strains that are isogenic except for capsular type. Serotype 4 strain TIGR4 was rendered unencapsulated by recombinational replacement of the capsular polysaccharide synthesis (cps) locus with the bicistronic Janus cassette (C. K. Sung, J. P. Claverys, and D. A. Morrison, Appl. Environ. Microbiol. 67:5190-5196, 2001). In subsequent transformation with chromosomal DNA, the cassette was replaced by the cps locus derived from a strain of a different serotype, either 6B, 7F, 14, or 19F. To minimize the risk of uncontrolled recombinational replacements in loci other than cps, the TIGRcps::Janus strain was "backcross" transformed three times with chromosomal DNA of subsequently constructed capsular type transformants. Capsular serotypes were confirmed in all new capsule variants by the Quellung reaction. Restriction fragment length polymorphism (RFLP) analysis of the cps locus confirmed the integrity of the cps region transformed into the TIGR strain, and RFLP of the flanking regions confirmed their identities with the corresponding regions of the recipient. Transformants had in vitro growth rates greater than or equal to that of TIGR4. All four strains were able to colonize C57BL/6 mice (female, 6 weeks old) for at least 7 days when mice were intranasally inoculated with 6 x 10(6) to 8 x 10(6) CFU. The constructed capsular variants of TIGR4 are suitable for use in studies on the role of S. pneumoniae capsular polysaccharide in immunity, colonization, and pathogenesis.     

Methodologies for Salmonella enterica subsp. enterica subtyping: gold standards and alternatives

For more than 80 years, subtyping of Salmonella enterica has been routinely performed by serotyping, a method in which surface antigens are identified based on agglutination reactions with specific antibodies. The serotyping scheme, which is continuously updated as new serovars are discovered, has generated over time a data set of the utmost significance, allowing long-term epidemiological surveillance of Salmonella in the food chain and in public health control. Conceptually, serotyping provides no information regarding the phyletic relationships inside the different Salmonella enterica subspecies. In epidemiological investigations, identification and tracking of salmonellosis outbreaks require the use of methods that can fingerprint the causative strains at a taxonomic level far more specific than the one achieved by serotyping. During the last 2 decades, alternative methods that could successfully identify the serovar of a given strain by probing its DNA have emerged, and molecular biology-based methods have been made available to address phylogeny and fingerprinting issues. At the same time, accredited diagnostics have become increasingly generalized, imposing stringent methodological requirements in terms of traceability and measurability. In these new contexts, the hand-crafted character of classical serotyping is being challenged, although it is widely accepted that classification into serovars should be maintained. This review summarizes and discusses modern typing methods, with a particular focus on those having potential as alternatives for classical serotyping or for subtyping Salmonella strains at a deeper level.     

Development of rapid coagulase serotyping method by PCR and microplate hybridization

We developed a novel PCR-based method for coagulase serotyping. Coagulase gene amplicons biotinylated by PCR were identified by microplate hybridization (MPH) using serotype-specific probes. The conventional serotyping method, which is strongly dependent on coagulase activity, may sometimes give a mistaken determination of the serotype, especially in cases where there is high coagulase activity. In contrast, the results of PCR-MPH are not affected by coagulase activity. Furthermore, once the isolated colonies are obtained, it only takes 3 h to perform PCR-MPH, and the interpretation of the results is entirely objective. We compared PCR-MPH with the conventional method for 90 strains of coagulase-producing Staphylococcus aureus. The same results were found for both the PCR-MPH and conventional methods, and thus, our results indicate that PCR-MPH is a rapid, objective, and reliable method for coagulase serotyping.     

In vitro studies on the degradation of poly(cis‐1,4‐isoprene)

Cleavage of the backbone of poly(cis‐1,4‐isoprene) (IR) in solid rubber material was accomplished by the addition of partially purified latex clearing protein (Lcp1_VH2) using a 200‐mL enzyme reactor. Two strategies for the addition of Lcp1_VH2 were studied revealing that the daily addition of 50 µg mL^?1 of Lcp1_VH2 for 5 days was clearly a more efficient regime in comparison to a one‐time addition of 250 µg of Lcp1_VH2 at the beginning. Soluble oligo(cis‐1,4‐isoprene) molecules occurred as degradation products and were identified by ESI‐MS and GPC. Oxygenase activity of Lcp1_VH2 with solid IR particles as substrate was shown for the first time by measuring the oxygen consumption in the reaction medium. A strong decrease of the dissolved oxygen concentration was detected at the end of the assay, which indicates an increase in the number of cleavage reactions. The oligo(cis‐1,4‐isoprene) molecules comprised 1 to 11 isoprene units and exhibited an average molecular weight (M_n) of 885 g mol^?1. Isolation of the oligo(cis‐1,4‐isoprene) molecules was achieved by using silica gel column chromatography. The relative quantification of the isolated products was performed by HPLC‐MS after derivatization with 2,4‐dinitrophenilhydrazyne yielding a concentration of total degradation products of 1.62 g L^?1. Analysis of the polymer surface in samples incubated for 3 days with Lcp1_VH2 via ATR‐FTIR indicated the presence of carbonyl groups, which occurred upon the cleavage reaction. This study presents a cell‐free bioprocess as an alternative rubber treatment that can be applied for the partial degradation of the polymer. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:890–899, 2018     

Realtime PCR Is More Sensitive than Multiplex PCR for Diagnosis and Serotyping in Children with Culture Negative Pneumococcal Invasive Disease

Background

Pneumococcal serotyping is usually performed by Quellung reaction, considered the gold standard test. However the method cannot be used on culture-negative samples. Molecular methods can be a useful alternative. The aim of the study was to evaluate the use of Multiplex-sequential-PCR (MS-PCR) or Realtime-PCR on blood samples for diagnosis and serotyping of invasive pneumococcal disease (IPD) in a pediatric clinical setting.

Methodology/Principal Findings

Sensitivity and specificity of MS-PCR and Realtime-PCR have been evaluated both on 46 well characterized pneumococcal isolates and on 67 clinical samples from children with culture-negative IPD. No difference in sensitivity and specificity between MS-PCR and Realtime PCR was found when the methods were used on isolates: both methods could type 100% isolates and the results were always consistent with culture-based methods. On the contrary, when used on clinical samples 43/67 (64.2%) were typeable by MS-PCR and 61/67 (91.0%) by Realtime-PCR (p = 0.0004,K Cohen 0.3, McNemar''s p<0.001). Non-typeability by MS-PCR was associated in 18/20 cases (90.0%) with low bacterial load. The difference between the two methods was present both when they were used on normally sterile fluids (respectively 31/33 (93.9%) typeable samples for Realtime-PCR and 24/33 (72.7%) for MS-PCR, p = 0.047, 95%CL 0.03–0.98; K Cohen 0.3; McNemar''s p = 0.0016) and when they were used on nasopharyngeal swabs (respectively 30/34 (88.2%) typeable samples for Realtime-PCR and 19/34 (55.9%) for MS-PCR, p = 0.007, 95%CL 0.04–0.66); the presence of multiple pneumococcal serotypes in nasopharyngeal swabs was found more frequently by Realtime PCR (19/30; 63.3%) than by Multiplex-sequential PCR (3/19; 15.8%; p = 0.003;95%CL 1.87–39.97).

Conclusions/Significance

In conclusion, both MS-PCR and Realtime PCR can be used for pneumococcal serotyping of most serotypes/serogroups directly on clinical samples from culture-negative patients but Realtime-PCR appears more sensitive.     

Surface antigen structure of Neisseria meningitidis. I. The isolation of a serotypic antigenic complex of N. meningitidis strain B16B6 by direct detergent treatment and its immunochemical characteristics

The method for obtaining a serotyping antigenic complex from N. meningitidis B16B6 by their direct treatment with the mixture of detergents (0.5% sodium desoxycholate and 0,5% cholic acid in the proportion 1 : 1) in 0.5 M KCl solution is proposed. Such treatment has been found to increase the yield of the preparation in terms of protein more than 4 times in comparison with earlier methods for obtaining serotyping antigens. The immunochemical study of the preparation has demonstrated its serological specificity and high immunological activity, not inferior to that of serotyping antigenic preparations from group B meningococci, obtained by the heretofore known methods.     

Single-step capsular transformation and acquisition of penicillin resistance in Streptococcus pneumoniae

The capsule (cps) locus of Streptococcus pneumoniae is flanked by the pbp2x and pbp1a genes, coding for penicillin-binding proteins, enzymes involved in cell wall synthesis that are targets for beta-lactams. This linkage suggested to us that selection for beta-lactam resistance might coselect for capsular transformants. The recombination event would then involve PBP genes, as well as the cps operon, and would change both the serotype and the resistance profile of the strain. We transformed beta-lactam-susceptible strain TIGR4 by using whole genomic DNA extracted from multidrug-resistant strain GA71, a serotype 19F variant of pneumococcal clone Spain(23F)-1, and selected beta-lactam-resistant transformants. Smooth colonies appearing on selective plates were subcultured, serotyped by the Quellung reaction, and genotyped to confirm the presence of the GA71 pbp2x-cps19-pbp1a locus in the TIGR4 genetic background by restriction fragment length polymorphism analysis of the whole locus and its flanking regions. The results showed that a new serotype, combined with resistance to beta-lactams, could emerge in a susceptible strain via a single transformation event. Quantitative analysis showed that transfer of the cps locus had occurred at an elevated rate in beta-lactam-selected transformants. This suggests that in natural settings selection by host immunity and selection by antibiotics may be interrelated because of "hitchhiking" effects due to linkage of resistance determinants and the capsule locus.     

The Use of Polyvalent Sera for the Serotyping of Mycobacteria within the Mycobacterium avium– M. intracellulare-M. scrofulaceum Complex

A modification to Schaefer's agglutination method for serotyping mycobacteria within the Mycobacterium avium-M. intracellulare-M. scrofulaceum complex is described. The antigens are screened against polyvalent sera and subsequently a reduced range of absorbed antisera. This expedites the serotyping procedure and conserves expensive antiserum stocks.     

pep27 and lytA in Vancomycin-Tolerant Pneumococci

Vancomycin therapy failure due to the emergence of tolerance in pneumococci is increasing. The molecular mechanism of tolerance is not clear, but lytA and pep27 are known to be involved. Our aim was to evaluate the expression of both genes in vancomycin-tolerant Streptococcus pneumoniae (VTSP) strains. Eleven VTSP strains from a total of 309 clinical isolates of S. pneumoniae from 1997 to 2006 were classified according to the criteria of Liu and Tomasz. All VTSP strains were evaluated for susceptibility according to CLSI criteria, serotype by the Quellung test, and clonality by PFGE. The expressions of lytA and pep27 were analyzed in different growth phases by RT-PCR with and without vancomycin. Eighty-two percent of VTSP strains showed resistance to penicillin, and 100% were sensitive to vancomycin and cefotaxime. The most frequent serotypes of VTSP strains were 23F (4/11) and 6B (3/11). Clonal relationship was observed in only two strains. No significant changes were observed in pep27 expression in the three phases of growth in VTSP strains with and without vancomycin. Interestingly, pep27 expression in the stationary phase in the non-tolerant reference strain R6 was significantly higher. However, no significant differences in lytA expression were observed between VTSP and R6 strains during the phases of growth analyzed. The absence of changes in pep27 expression in VTSP strains in the stationary phase may be related to their ability to tolerate high antibiotic concentrations, and thus, they survive and remain in the host under the antibiotic selective pressure reflected in therapeutic failure.     

Classification of Salmonella enterica serotypes from Australian poultry using repetitive sequence-based PCR

Aims: To evaluate a semi‐automated repetitive extragenic palindromic sequence‐based PCR (rep‐PCR) system for the classification of Salmonella serotypes from Australian poultry. Methods and Results: Using a DNA fingerprint library within the DiversiLab^® System, four separate databases were constructed (serogroup B, C, E and Other). These databases contained 483 serologically confirmed (reference laboratory) Salmonella isolates. A blinded set of Salmonella cultures (n = 155) were typed by rep‐PCR, matched against the internal library and compared with traditional serotyping. The predicted (Kullback–Leibler) serotype of 143 (92·3%) isolates matched traditional typing (P < 0·05). Of the 12 (7·7%) remaining isolates, ten (6·5%) resulted in ‘No Match’, one (0·65%) was incorrectly matched to the library (Salm. subsp 1 ser 4,12:‐:‐), and the other (0·65%) was referenced as Salm. ser. Sofia, whereas rep‐PCR and in‐house serotyping concurred as Salmonella serovar Typhimurium. Financial analysis showed higher material cost (215%) and a lower labour component (47·5%) for rep‐PCR compared with serotyping. Conclusion: The DiversiLab^® System, with serogroup databases, was successfully implemented as an adjunct for reference serotyping of Salmonella enterica. Significance and Impact of the Study: The DiversiLab^® System platform is a cost‐effective and easy‐to‐use system, which can putatively determine Salmonella enterica serotypes within a few hours.     

Combined sigB allelic typing and multiplex PCR provide improved discriminatory power and reliability for Listeria monocytogenes molecular serotyping

Conventional serotyping has traditionally been used to subtype Listeria monocytogenes, but has several limitations, including low discriminatory power and poor reproducibility. Molecular serotyping methods have been developed for L. monocytogenes, but generally show limited discriminatory power and high misclassification rates. We selected 157 Listeria isolates to evaluate a combination of a previously described multiplex PCR assay and sigB allelic typing as an alternative molecular serotyping and subtyping strategy for L. monocytogenes. While the multiplex PCR assay differentiated five L. monocytogenes subtypes (Simpson's Index of Discrimination [SID]=0.78), including classification of the most common disease-associated serotypes (1/2a, 1/2b, 1/2c, and lineage I 4b) into four distinct groups, it misclassified 3.8% of the isolates studied here. sigB allelic typing differentiated 29 subtypes (SID=0.87) and also allowed identification of lineage III L. monocytogenes, which could not be differentiated from the other Listeria spp. by the multiplex PCR assay. sigB allelic typing failed to differentiate serotype 1/2c and 1/2a isolates and one sigB allelic type included serotype 4b and 1/2b isolates. A molecular serotyping approach that combines multiplex PCR and sigB sequence data showed increased discriminatory power (SID=0.91) over either method alone as well as conventional serotyping (SID=0.87) and classifies the four major serotypes (i.e., 1/2a, 1/2b, 1/2c, and 4b) into unique subgroups with a lower misclassification rate as compared to the multiplex PCR assay. This combined approach also differentiates lineage I serotype 4b isolates from the genetically distinct serotype 4b isolates classified into lineage III.