Traditional Serotyping Research Articles

Molecular Surveillance of Clinical Neisseria gonorrhoeae Isolates in Russia

The choice of adequate methods for epidemiological purposes remains a challenging problem in Neisseria gonorrhoeae molecular monitoring. In this study, the collection of geographically unrelated gonococci (n = 103) isolated in Russian clinics was comparably tested by (i) a traditional serotyping scheme, (ii) por typing, (iii) Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST), and (iv) multilocus sequence typing (MLST). It is shown that, according to sequencing data, a third of the strains carried new porB1 alleles, as well as tbpB ones, and more than half of the samples had new sequence types (STs) as determined by NG-MAST or MLST. The discriminatory power for each typing method was calculated by using the Hunter-Gaston discriminatory index, D. Commonly, modern nucleic acid-based typing methods (por typing, NG-MAST, and MLST) appeared to be more efficient than the classical serotyping scheme. While the traditional serotyping gave a D value of 0.82, the por typing, NG-MAST, and MLST approaches yielded D values of 0.97, 0.98, and 0.91, respectively. Each typing technique revealed the distribution of gonococci slightly correlated with their geographical sources. However, only the MLST method STs were highly associated with certain phenotypes. Although ST1594, ST1892, and ST6720 were typical for susceptible gonococci, ST1901 and ST6716 were undoubtedly associated with a multidrug-resistant phenotype. We conclude that every tested nucleic acid-based typing method is suitable for N. gonorrhoeae molecular surveillance. However, the MLST method seems to serve large-scale epidemiological purposes, whereas the NG-MAST and por typing approaches are more appropriate for the investigation of local outbreaks.

Development and Evaluation of a Multiplex Real-Time Polymerase Chain Reaction Procedure to Clinically Type Prevalent Salmonella enterica Serovars

A multiplex real-time polymerase chain reaction procedure was developed to identify the most prevalent clinical isolates of Salmonella enterica subsp. enterica. Genes from the rfb, fliC, fljB, and viaB groups that encode the O, H, and Vi antigens were used to design 15 primer pairs and TaqMan probes specific for the genes rfbJ, wzx, fliC, fljB, wcdB, the sdf-l sequence, and invA, which was used as an internal amplification control. The primers and probes were variously combined into six sets. The first round of reactions used two of these sets to detect Salmonella O:4, O:9, O:7, O:8, and O:3,10 serogroups. Once the serogroups were identified, the results of a second round of reactions that used primers and probes for the flagellar antigen l genes, 1,2; e,h; g,m; d; e,n,x; and z(10), and the Vi gene were used to identify individual serovars. The procedure was standardized using 18 Salmonella reference strains and other enterobacteria. The procedure's reliability and sensitivity was evaluated using 267 randomly chosen serotyped Salmonella clinical isolates. The procedure had a sensitivity of 95.5% and was 100% specific. Thus, our technique is a quick, sensitive, reliable, and specific means of identifying S. enterica serovars and can be used in conjunction with traditional serotyping. Other primer and probe combinations could be used to increase the number of identifiable serovars.

Characterization of the Salmonella Isolates from Backyard Chickens in North of Iran, by Serotyping, Multiplex PCR and Antibiotic Resistance Analysis

The present study was designed to investigate the prevalence of Salmonella species, their molecular characterization and antibiotic resistance in backyard chickens. A total of 1125 samples were collected from backyard chickens in two consequent samplings. In the first part, samples were included of 820 poor recently hatched chicks, hatching residuals, egg shells in the nest floor, cloacal swabs and fresh litter droppings in the villages which located in north of Iran. Secondly, 305 samples were taken from newly hatched-chicks which fertile eggs were obtained from the rural chickens of those regions and incubated in laboratory incubator. Of 1125 samples tested, 27 (2.4 %) Salmonella were isolated that identified as serovars of Salmonella enteritidis (55.5 %), Salmonella typhimurium (22.2 %), Salmonella hadar (14.8 %) and Salmonella infantis (7.4 %). Except the traditional serotyping that was performed for all isolates, Salmonella typhimurium and Salmonella enteritidis isolates were characterized by using multiplex PCR for further identification. All of six Typhimurium serovars were positive for rfbJ, fljB, invA and fliC genes. In the case of Enteritidis serovars, polymerase chain reaction generated amplification products for spv, sefA and random sequence (specific for the genus Salmonella) in all of fifteen samples. Most of the Salmonella isolates in this study were sensitive to norfloxacin.

Identification of 20 Common Human Enterovirus Serotypes by Use of a Reverse Transcription-PCR-Based Reverse Line Blot Hybridization Assay

The more than 100 human enterovirus (HEV) serotypes can also be classified into four species, HEV-A to -D, based on phylogenetic analysis of multiple gene regions. Current molecular typing methods depend largely on reverse transcription-PCR (RT-PCR) amplification and nucleotide sequencing of the entire or 3' half of the VP1 gene. An RT-PCR-based reverse line blot (RLB) hybridization assay was developed as a rapid and efficient approach to characterize common HEVs. Twenty HEV serotypes accounted for 87.1% of all HEVs isolated at an Australian reference virology laboratory from 1979 to 2007. VP1 sequences of all known HEV prototype strains were aligned to design one sense primer and three antisense primers for RT-PCR. After sequencing of the complete VP1 genes of 37 previously serotyped examples of the commonest 20 serotypes and alignment of these VP1 sequences with GenBank sequences, four serotype-specific probes for each serotype were designed for RLB. The RT-PCR-RLB assay was then applied to 132 HEV isolates, made up of the previously sequenced 37 isolates and another 95 serotyped clinical isolates. The RT-PCR-RLB genotypes corresponded with the serotypes for 131/132 isolates; the one exception was confirmed by VP1 sequencing, and the genotype was confirmed by repeat conventional serotyping. Genotyping by RT-PCR-RLB complements traditional serotyping methods and VP1 sequencing and has the advantages of convenience, speed, and accuracy. RT-PCR-RLB allows detection of specific enteroviral serotypes or genotypes associated with HEV outbreaks and significant disease.

Development of an oligonucleotide microarray method for Salmonella serotyping

SummaryAdequate identification of Salmonella enterica serovars is a prerequisite for any epidemiological investigation. This is traditionally obtained via a combination of biochemical and serological typing. However, primary strain isolation and traditional serotyping is time‐consuming and faster methods would be desirable. A microarray, based on two housekeeping and two virulence marker genes (atpD, gyrB, fliC and fljB), has been developed for the detection and identification of the two species of Salmonella (S. enterica and S. bongori), the five subspecies of S. enterica (II, IIIa, IIIb, IV, VI) and 43 S. enterica ssp. enterica serovars (covering the most prevalent ones in Austria and the UK). A comprehensive set of probes (n = 240), forming 119 probe units, was developed based on the corresponding sequences of 148 Salmonella strains, successfully validated with 57 Salmonella strains and subsequently evaluated with 35 blind samples including isolated serotypes and mixtures of different serotypes. Results demonstrated a strong discriminatory ability of the microarray among Salmonella serovars. Threshold for detection was 1 colony forming unit per 25 g of food sample following overnight (14 h) enrichment.

Capsular Polysaccharide Synthesis Regions in Klebsiella pneumoniae Serotype K57 and a New Capsular Serotype

Community-acquired pyogenic liver abscess caused by Klebsiella pneumoniae is an emerging infectious disease. We explored the capsular polysaccharide synthesis (cps) regions of three non-K1, non-K2 K. pneumoniae strains, A1142, A7754, and A1517, from Taiwanese patients experiencing pyogenic liver abscess. Two of the strains, A1142 and A7754, belonged to capsular serotype K57, while the third belonged to a new capsular serotype, different from the previously reported 77 serotypes. Deletion and complementation experiments suggested that a unique K57 gene, a homologue of wzy, was essential for K57 capsular synthesis and confirmed that this gene cluster was a genetic coding region for K57. Compared to K1 and K2 strains, the three strains were all serum sensitive, suggesting that host factors might also be involved in the three patients. PCR using primers from specific genes for K57 was more sensitive and specific than traditional serotyping. The remaining strain, A1517, did not react to the antisera from any of the 77 serotypes, and none of the 77 reference strains reacted to the serum against this strain. Moreover, PCR analyses using various primer pairs from the serotype-specific open reading frames did not reveal cross-reactivity to any of the 77 reference strains, suggesting that this strain likely represents a new capsular type. We conclude that sequences from these two cps regions are very useful in detecting K57 and the new cps genotype.

Development of a multiplex PCR test for identification of Actinobacillus pleuropneumoniae serovars 1, 7, and 12

A PCR assay for simultaneous species identification and separation of Actinobacillus pleuropneumoniae serovars 1, 7 and 12 was developed. Primers specific for genes involved in biosynthesis of the capsular polysaccharides ( cps genes) of serovars 1, 7, and 12 were combined with a species-specific PCR test based on the omlA gene. The PCR test was evaluated with the serovar reference strains of A. pleuropneumoniae as well as 183 Danish field isolates. For all typable strains, a complete correspondence was found between results obtained with the multiplex PCR test and results from the traditional serotyping methods. Among eight serologically cross-reacting strains designated K1:O7, seven isolates produced amplicons of similar sizes as serovar 1 and one isolate produced amplicons of similar sizes as serovar 7. The species specificity of the assay was evaluated using a collection of 126 strains representing 25 different species within the family Pasteurellaceae including 45 field strains of the phylogenetically affiliated species Actinobacillus lignieresii. All these isolates tested negative for the cps genes by the multiplex PCR test except for 6 isolates of A. lignieresii. Five of these isolates produced an amplicon identical to the cps gene of serovar 7, whereas one isolate produced an amplicon identical to the cps gene of serovar 1. In addition, four isolates of Actinobacillus genomospecies 1 tested positive for the omlA gene but negative for the cps genes. The test represents a convenient and specific method for serotyping A. pleuropneumoniae in diagnostic laboratories.

Novel PCR assay for identification of Salmonella enterica serovar Infantis

We developed, optimized and tested two novel PCR assays specific for Salmonella enterica subspecies enterica serovar Infantis. The fljB gene was chosen as the target sequence. Primers were designed on a consensus sequence built by sequencing the fljB gene of five genetically unrelated Hungarian S. Infantis strains and using sequence data from the GenBank (http://www.ncbi.nih.gov). Two alternative assays were designed, which share the reverse primer. Both proved to be highly specific to S. Infantis, neither reacted with 42 other nontyphoidal serovariants tested. The detection limit of the assays was determined to be 10(5) CFU ml(-1) from pure culture, and 10(6) CFU g(-1) from artificially spiked chicken faeces samples. Although the detection limit is rather high to allow for using them for direct detection, the assays may be useful in identification of S. Infantis both for diagnostic and for research purposes. The described PCR assays allow for the correct identification of S. Infantis even when traditional serotyping methods fail because lack of expression of flagellar antigens.

Multiplex, Bead-Based Suspension Array for Molecular Determination of Common Salmonella Serogroups

We report the development and evaluation of a Salmonella O-group-specific Bio-Plex assay to detect the six most common serogroups in the United States (B, C(1), C(2), D, E, and O13) plus serotype Paratyphi A. The assay is based on rfb gene targets directly involved in O-antigen biosynthesis; it can be completed 45 min post-PCR amplification. The assay correctly and specifically identified 362 of 384 (94.3%) isolates tested in comparison to traditional serotyping. Seventeen isolates (4.4%) produced results consistent with what is known about the molecular basis for serotypes but different from the results of traditional serotyping, and five isolates (1.3%) generated false-negative results. Molecular determination of the serogroup for rough isolates was consistent with a common serotype in most instances, indicating that this approach has the potential to provide O-group information for isolates that do not express an O antigen. We also report the sequence of the O-antigen-encoding rfb gene cluster from Salmonella enterica serotype Poona (serogroup O13). Compared with other, previously characterized rfb regions, the O13 rfb gene cluster was most closely related to Escherichia coli O127 and O86. The O-group Bio-Plex assay described here provides an easy-to-use, high-throughput system for rapid detection of common Salmonella serogroups.

A real-time PCR for the detection of Salmonella Enteritidis in poultry meat and consumption eggs

A robust duplex 5′ nuclease (TaqMan) real-time PCR was developed and in-house validated for the specific detection of Salmonella enterica subspecies enterica serovar Enteritidis in whole chicken carcass rinses and consumption eggs. The assay uses specifically designed primers and a TaqMan probe to target the Prot6e gene located on the S. Enteritidis specific 60-kb virulence plasmid. As an internal amplification control to monitor Salmonella DNA in the sample, a second primer/TaqMan probe set detects simultaneously the Salmonella specific invA gene. The assay identified correctly 95% of the 79 Salmonella Enteritidis strains tested comprising 19 different phage types. None of the 119 non-Enteritidis strains comprising 54 serovars was positive for the Prot6e gene. The assay detection probability was for 10 2 or more genome equivalents 100% and for 10 equivalents 83%. A pre-PCR sample preparation protocol including a pre-enrichment step in buffered peptone water, followed by DNA extraction was applied on low levels of artificially contaminated whole chicken carcass rinses and eggs from hens as well as 25 potentially naturally contaminated chickens. The detection limit was less than three CFU per 50 ml carcass rinse or 10 ml egg. The sensitivity and specificity compared to the traditional culture-based detection method and serotyping were both 100%. Twenty-five potentially naturally contaminated chickens were compared by the real-time PCR and the traditional cultural isolation method resulting in four Salmonella positive samples of which two were positive for the Prot6e gene and serotyped as S. Enteritidis. We show also that Salmonella isolates which have a rough lipopolysaccharide structure could be assigned to the serovar Enteritidis by the real-time PCR. This methodology can contribute to meet the need of fast identification and detection methods for use in monitoring and control measures programmes.

Molecular serotyping of Escherichia coli O111:H8

Accurate Escherichia coli serotyping is critical for pathogen diagnosis and surveillance of non-O157 Shiga-toxigenic strains, however few laboratories have this capacity. The molecular serotyping protocol described in this paper targets the somatic and flagellar antigens of E. coli O111:H8 used in traditional serotyping, and can be performed routinely in the laboratory.

Blind comparison of traditional serotyping with three multiplex PCRs for the identification of Salmonella serotypes

Salmonella serotypes are defined on the basis of somatic (O) antigens which define the serogroup and flagellar (H) factor antigens, both of which are present in the cell wall of Salmonella. Most Salmonella organisms alternatively express phase-1 or phase-2 flagellar antigens encoded by fliC and fljB genes, respectively. Our group previously published two multiplex PCRs for distinguishing the most common first- and second-phase antigens. In this paper we describe a third multiplex PCR to identify the most common serogroups (O:B; O:C1; O:C2; O:D and O:E). The combination of these three PCRs enabled us to completely serotype organisms belonging to the Salmonella species. This multiplex PCR includes 10 primers. A total of 67 Salmonella strains belonging to 32 different serotypes were tested. Each strain generated one serogroup-specific fragment ranging between 162 and 615 bp. Twenty-eight strains belonging to 21 serotypes, with a serogroup different from those tested in this work, did not generate any fragments. To compare molecular serotyping with traditional serotyping, 500 strains, received according to the order of arrival in the laboratory, were serotyped using both methods. The three multiplex PCRs were able to serotype 84.6% of the tested strains. This method was found to be very helpful in our laboratory as an alternative method for typing strains causing outbreaks, and it can be used to supplement conventional serotyping, since it is also applicable to motionless and rough strains.

Use of a Serotype-Specific DNA Microarray for Identification of Group B Streptococcus ( Streptococcus agalactiae )

Group B Streptococcus (GBS; Streptococcus agalactiae) is an important cause of sepsis and meningitis. Nine GBS serotypes, based on capsular polysaccharide (CPS) antigens, have been described. Their distribution varies worldwide and needs to be monitored to understand the epidemiology of GBS disease and inform the development of vaccines. In this study, we sequenced cpsH of GBS serotype II (cpsHII) and compared it with that of the other eight serotypes to identify serotype-specific regions. We then developed a DNA microarray based on the cpsH gene and used it to test 88 GBS isolates-9 serotype reference strains and 79 clinical isolates-and 7 other bacterial and fungal species which are commonly present in the vagina flora. The microarray was shown to be specific and reproducible. This is the first report of a microarray which can identify the nine GBS serotypes. The use of a microarray has advantages over traditional serotyping methods and will be of practical value in both reference and diagnostic laboratories.

Discrimination of Listeria monocytogenes contaminated commercial Japanese meats

Discrimination was attempted on 14 Listeria monocytogenes strains isolated from commercially available Japanese pork and chicken. Examination of the isolates was performed by restriction fragment length polymorphism (RFLP) analysis of the chromosomal DNA and amplified products and comparison of the nucleotide sequences of the amplified products. A polymorphism region containing the repeated sequences in the iap gene was amplified by the polymerase chain reaction (PCR). The genetic analyses could discriminate the 14 isolates in combination with traditional serotyping, and some strains isolated from different meats were confirmed to have a genetically close relationship. Genetic analyses used in the present study would be useful for the elucidation of the pathogen tracks from contaminated sources to humans and of the ecological niche in the food environment.

Molecular Serotyping ofEscherichia coliO26:H11

Serotyping is the foundation of pathogenic Escherichia coli diagnostics; however, few laboratories have this capacity. We developed a molecular serotyping protocol that targets, genetically, the same somatic and flagellar antigens of E. coli O26:H11 used in traditional serotyping. It correctly serotypes strains untypeable by traditional methods, affording primary laboratories serotyping capabilities.

Molecular epidemiology of group B streptococcal infections

Streptococcus agalactiae (GBS) is a causative agent of sepsis and meningitis in newborns and diseases in pregnant women and nonpregnant adults. Various approaches, including both nongenetic and genetic techniques, are currently used for the study of epidemiology of GBS infections. In the present paper the different methods of molecular epidemiology of GBS infections are reviewed, and several novel approaches are introduced. The advantages and disadvantages of molecular methods are discussed and compared with traditional serotyping technique. The possible use of the molecular approaches for identification of different genetic lineages in GBS as well as for identification and control of the epidemiologically actual clones is discussed.

Towards the development of a DNA-sequence based approach to serotyping of Salmonella enterica

BackgroundThe fliC and fljB genes in Salmonella code for the phase 1 (H1) and phase 2 (H2) flagellin respectively, the rfb cluster encodes the majority of enzymes for polysaccharide (O) antigen biosynthesis, together they determine the antigenic profile by which Salmonella are identified. Sequencing and characterisation of fliC was performed in the development of a molecular serotyping technique.ResultsFliC sequencing of 106 strains revealed two groups; the g-complex included those exhibiting "g" or "m,t" antigenic factors, and the non-g strains which formed a second more diverse group. Variation in fliC was characterised and sero-specific motifs identified. Furthermore, it was possible to identify differences in certain H antigens that are not detected by traditional serotyping. A rapid short sequencing assay was developed to target serotype-specific sequence motifs in fliC. The assay was evaluated for identification of H1 antigens with a panel of 55 strains.ConclusionFliC sequences were obtained for more than 100 strains comprising 29 different H1 alleles. Unique pyrosequencing profiles corresponding to the H1 component of the serotype were generated reproducibly for the 23 alleles represented in the evaluation panel. Short read sequence assays can now be used to identify fliC alleles in approximately 97% of the 50 medically most important Salmonella in England and Wales. Capability for high throughput testing and automation give these assays considerable advantages over traditional methods.

Evaluation of a multiplex PCR test for simultaneous identification and serotyping of Actinobacillus pleuropneumoniae serotypes 2, 5, and 6.

Serotype-specific DNA regions involved in the biosynthesis of capsular polysaccharides (cps region) were used to develop a multiplex PCR test for the simultaneous species identification and serotyping of Actinobacillus pleuropneumoniae serotypes 2, 5, and 6. Primers specific for serotypes 2, 5, and 6 were combined with the already existing species-specific primers used in a PCR test based on the omlA gene. The PCR test was evaluated with serotype reference strains of A. pleuropneumoniae as well as 182 Danish field isolates previously serotyped by latex agglutination or immunodiffusion. For all serologically typeable strains, a complete correspondence was found between the results obtained by the multiplex PCR test and the results obtained by the traditional serotyping methods. Six of eight serologically nontypeable strains could be allocated to a serotype on the basis of the multiplex PCR results. The species specificity of the assay was evaluated with a collection of 93 strains representing 29 different species within the family Pasteurellaceae, as well as species normally found in the respiratory tracts of swine. All of these strains were negative by the multiplex PCR test, including 50 field isolates of the phylogenetically closely related species Actinobacillus lignieresii. When the multiplex PCR test was used to test Danish field strains, it was able to identify the serotypes of approximately 94% of all strains isolated from swine with clinical disease. More than 90% of the isolates that cross-reacted by the latex agglutination test were of serotype 2, 5, or 6. Determination of the serotype by PCR represents a convenient and specific method for the serotyping of A. pleuropneumoniae in diagnostic laboratories.

Serotyping and Ribotyping of Salmonella Using Restriction Enzyme PvuII

The subtyping and identification of bacterial pathogens throughout food processing and production chains is useful to the new hazard analysis critical control point–based food safety plans. Traditional manual serotyping remains the primary means of subtyping Salmonella isolates. Molecular biology techniques, however, offer the promise of more rapid and sensitive subtyping of Salmonella. This study evaluates the potential of restriction enzyme PvuII, followed by probing with the rRNA operon from Escherichia coli, to generate serotype-specific DNA fingerprints. A total of 32 identified serotypes were found with an overall agreement in 208 of the 259 (80%) isolates tested between U.S. Department of Agriculture serotype identification and riboprint serotype identification. Many of the isolates that did not correlate were serotype identified as Salmonella Montevideo, which indicates that for this serotype, there are multiple ribotypes. When Salmonella Montevideo isolates were not included, the ribotype identification agreed with serotyping in 207 of the 231 (90%) isolates. The primary outcome of any ribotyping procedure is to give distinct ribotype patterns. This extensive poultry epidemiological study demonstrates that, in addition to ribotype patterns, the identification of isolates to known serotypes provides the investigator with additional information that can be more useful than traditional epidemiology and isolate identification studies.

Fowl cholera.

Pasteurella multocida subspecies multocida is the most common cause of fowl cholera, although P. multocida subspecies septica and gallicida may also cause fowl cholera-like disease to some extent. However, the virulence properties of the different subspecies for various hosts have not been elucidated. The severity and incidence of P. multocida infections may vary considerably depending on several factors associated with the host (including species and age of infected birds), the environment and the bacterial strain. No single virulence factor has been associated with the observed variation in virulence among strains. Possible virulence factors include the following: the capsule, endotoxin, outer membrane proteins, iron binding systems, heat shock proteins, neuraminidase production and antibody cleaving enzymes. No RTX toxins (repeats in toxin) appear to be produced by P. multocida, but P. multocida exotoxin (PMT) could contribute to virulence in some avian infections. The epidemiology of fowl cholera appears complex. Traditional serotyping systems are only of limited use in epidemiological studies. In recent years, molecular typing methods have been applied to avian strains of P. multocida of different origin. The results obtained using these newer methods indicate that wild birds may be a source of infection to commercial poultry. Documentation suggesting that mammals play a similar role is not as comprehensive, but the possibility cannot be excluded. Carrier birds seem to play a major role in the transmission of cholera. Surviving birds from diseased flocks appear to represent a risk, but more recent investigations indicate that carriers of P. multocida may exist within poultry flocks with no history of previous outbreaks of fowl cholera. The significance of this awaits further investigation. The site of infection for P. multocida is generally believed to be the respiratory tract. The outcome of infections may range from peracute/acute infections to chronic infections. In the former type of infections, few clinical signs are observed before death and the lesions will be dominated by general septicaemic lesions. In chronic forms of P. multocida infections, suppurative lesions may be widely distributed, often involving the respiratory tract, the conjunctiva and adjacent tissues of the head. Diagnosis is always dependent upon isolation of the organism. For the detection of subclinical infections, mouse passage of relevant samples is recommended, but polymerase chain reaction and isolation attempts on selective media may represent alternatives. Confinement is probably the most effective way to prevent introduction of P. multocida. However, extensive management systems dominate in many parts of the world, and under such circumstances vaccination is recommended as a preventive measure. Unfortunately, the development of safe and efficient live vaccines still poses problems. As a result, control remains dependent on bacterins which exhibit significant disadvantages compared to live vaccines.