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Choosing an appropriate bacterial typing technique for epidemiologic studies.

A wide variety of bacterial typing systems are currently in use that vary greatly with respect to the effort required, cost, reliability and ability to discriminate between bacterial strains. No one technique is optimal for all forms of investigation. We discuss the desired level of discrimination and need for a biologic basis for grouping strains of apparently different types when using bacterial typing techniques for different epidemiologic applications: 1) confirming epidemiologic linkage in outbreak investigations, 2) generating hypotheses about epidemiologic relationships between bacterial strains in the absence of epidemiologic information, and 3) describing the distributions of bacterial types and identifying determinants of those distributions. Inferences made from molecular epidemiologic studies of bacteria depend upon both the typing technique selected and the study design used; thus, choice of typing technique is pivotal for increasing our understanding of the pathogenesis and transmission, and eventual disease prevention.

Journal Article↗

An improved protocol for pulsed-field gel electrophoresis typing of Clostridium difficile.

Pulsed-field gel electrophoresis (PFGE) is the 'gold standard' technique for bacterial typing and has proved to be discriminatory and reproducible for typing Clostridium difficile. Nevertheless, a high proportion of strains are non-typable by this technique due to the degradation of the DNA during the process. The introduction of several modifications in the PFGE standard procedure increased typability from 40% (90 isolates) to 100% (220 isolates) while maintaining the high degree of discrimination and reproducibility of the technique.

Bacterial Typing Techniques↗

Transmission of periodontal bacteria and models of infection.

BACKGROUND: Bacteria play an essential role in the aetiology of periodontitis. Most bacterial species isolated from subgingival plaque are indigenous to the oral cavity. Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis are detected infrequently in periodontal health, which makes these species prime candidates to study person-to-person transmission. The aim of the present study was to review the literature on transmission of these periodontal bacterial species. METHOD: We review the literature on bacterial typing techniques and summarize the information on clonal distribution of A. actinomycetemcomitans and P. gingivalis in family units based on different typing techniques in order to establish the likelihood for person-to-person transmission of these periodontal pathogens. RESULTS: Vertical transmission of A. actinomycetemcomitans is estimated to be between 30% and 60%, whereas vertical transmission of P. gingivalis has rarely been observed. Horizontal transmission between spouses ranges between 14% and 60% for A. actinomycetemcomitans and between 30% and 75% for P. gingivalis. There is some evidence to show that cohabitation with a periodontitis patient influences the periodontal status of the spouse; however, substantially more information is needed to prove this hypothesis. CONCLUSIONS: Transmission of putative periodontal pathogens between family members has been shown. The clinical consequences of these events have been poorly documented. Based on the current knowledge, screening for and prevention of transmission of specific virulent clones of A. actinomycetemcomitans may be feasible and effective in preventing some forms of periodontal disease. P. gingivalis is usually recovered from diseased adult subjects, and transmission of this pathogens seems largely restricted to adult individuals. Horizontal transmission of P. gingivalis may therefore be controlled by periodontal treatment involving elimination or significant suppression of the pathogen in diseased individuals and by a high standard of oral hygiene.

Actinobacillus Infections↗

Use of polymerase chain reaction and specific monoclonal antibodies as rapid method to recognize Borrelia burgdorferi sensu stricto, B. garinii and B. afzelii among Italian isolates of B. burgdorferi.

We previously classified locally isolated strains of Borrelia burgdorferi by a restriction fragment length polymorphism analysis of total DNA, by DNA/DNA Southern Blot hybridization and by a hybridization with rRNA 16 + 23S from Escherichia coli [Cinco et al. (1993) Microbiologica 16:323-332] into three genetic groups which, according to the reference strains used, should correspond to the three species so far described as B. burgdorferi sensu stricto, B. garinii and B. afzelii. To find a simpler method for strain identification, in this study we analyzed the Italian strains and some strains identification, in this study we analyzed the Italian strains and some strains originating from other European countries, employing the species-specific 16S rRNA primers in the polymerase chain reaction technique (PCR) and some phenotypic markers like the B. afzelii-specific monoclonal antibodies and the battery of OspA-specific monoclonal antibodies which were reported to give a reactivity pattern correlated to the species [Wilske et al. (1993) J Clin Microbiol 31:340-350]. The PCR results confirmed those obtained previously by identifying the three groups as B. burgdorferi sensu stricto, B. garinii and B. afzelii; the reactivity patterns obtained with the monoclonal antibodies (mAb) also corresponded to those described as typical of the three species. We standardized the PCR technique to amplify a sample of crude template DNA obtained from a culture of 10(5) spirochetes.

Antibodies, Bacterial↗

Random amplified polymorphic DNA and amplified fragment length polymorphism analyses of Pasteurella multocida isolates from fatal fowl cholera infections.

Fowl cholera, a disease caused by Pasteurella multocida, continues to be a major problem for the poultry industry. The sources of pathogenic organisms responsible for most sporadic epidemics remain unconfirmed, although attenuated vaccines that retain a low level of virulence have occasionally been implicated in outbreaks of the disease. One of the vaccines most commonly used to prevent fowl cholera is the M-9 strain. In the present study, 61 clinical isolates from turkeys that died of fowl cholera from 1997 to 1999 on 36 Utah farms were analyzed and compared to the M-9 vaccine strain. Genetic analyses of the isolates were done by random amplified polymorphic DNA (RAPD) analysis and amplified fragment length polymorphism (AFLP) fingerprinting. The results of these genetic analyses were correlated with the vaccination status of the flock, isolate serotype, and geographic location. Although both genetic techniques effectively identified similar subtle genomic differences, RAPD analysis provided only 77% of the detail provided by AFLP analysis. While a relationship between genetic profile and serotype was evident, no significant relationship indicating geographic influence was found (P = 0.351). Interestingly, organisms isolated from vaccinated flocks were significantly closer genetically to the M-9 vaccine strain than isolates from unvaccinated birds were (P = 0.020). Statistical analyses revealed that this relationship could not have been determined by serotyping alone (P = 0.320), demonstrating the value of AFLP and RAPD analyses in the characterization of disease-causing strains.

Animals↗

An ospA-polymerase chain reaction/restriction fragment length polymorphism-based method for sensitive detection and reliable differentiation of all European Borrelia burgdorferi sensu lato species and OspA types.

We describe a sensitive and reliable method for detection and differentiation of the five relevant European Borrelia burgdorferi sensu lato species ( B. burgdorferi sensu stricto, B. afzelii, B. garinii, B. valaisiana, and B. lusitaniae), based on a heminested ospA-PCR followed by restriction enzyme analysis. Sensitivity was one borrelia per PCR except for B. afzelii, where it was five per PCR. None of seven relapsing fever borreliae, eight Leptospira serovars or two Treponema species were amplified. Except B. garinii, each of the five B. burgdorferi s.l. species is represented by one or two characteristic restriction fragment length polymorphism (RFLP) patterns. Analysis of the heterogeneous group of B. garinii resulted in five different RFLP patterns, corresponding to the OspA types 3-7 associated with this species. In a pilot study on 529 Ixodes ricinus ticks from three different regions in Southern Germany, all species and OspA types were found except B. lusitaniae and B. garinii OspA type 7, arguing for a broad distribution of almost all OspA types. A further notable finding was the focal prevalence of OspA type 4, which has rarely been detected in ticks previously. Thus, the developed method provides a fast and simple tool for epidemiological studies on the heterogeneity of species and OspA types in Europe which has important implications for the development of vaccines and (microbiological) test systems for Europe.

Animals↗

Construction and vaccine potential of an aroA mutant of Pasteurella haemolytica.

The aroA gene, encoding 5-enolpyruvylshikimate 3-phosphate synthase, from Pasteurella haemolytica biotype A, serotype 1 was cloned by complementation of the aroA mutation in Escherichia coli strain AB2829 after electroporation with a DNA library constructed in pUC18. The cloned P. haemolytica aroA gene was inactivated by insertion of a kanamycin resistance gene and reintroduced by allelic exchange into the chromosome of the parental P. haemolytica using PbluescriptII SK+. The P. haemolytica aroA mutant was highly attenuated in a mouse septicaemic model. Mice immunized intraperitoneally with two doses of live P. haemolytica aroA mutant were protected against a lethal parental strain challenge.

Animals↗

The identification and evaluation of ATP binding cassette systems in the intracellular bacterium Francisella tularensis.

Francisella tularensis is a facultative intracellular bacterium responsible for the disease tularemia. Analysis of the fully sequenced genome of the virulent F. tularensis strain SCHU S4 has led to the identification of twenty ATP binding cassette (ABC) systems, of which five appear to be non-functional. The fifteen complete systems comprise three importers, five exporters, four systems involved in non-transport processes, and three systems of unknown or ill-defined function. The number and classification of the ABC systems in F. tularensis is similar to that observed in other intracellular bacteria, indicating that some of these systems may be important for the intracellular lifestyle of these organisms. Among the ABC systems identified in the genome are systems that may be involved in the virulence of F. tularensis SCHU S4. Six ABC system proteins were evaluated as candidate vaccine antigens against tularemia, although none provided significant protection against F. tularensis. However, a greater understanding of these systems may lead to the development of countermeasures against F. tularensis.

ATP-Binding Cassette Transporters↗

Lipids of 'Mycobacterium habana', a synonym of Mycobacterium simiae with vaccine potential.

'Mycobacterium habana' was proposed as a distinct species within the genus Mycobacterium; however, it is actually a synonym of Mycobacterium simiae and included in the serotype I of this species. The potential use of 'M. habana' as a vaccine in both leprosy and tuberculosis has led to the analysis of its lipid composition in an attempt to define distinctive markers that could be used in the quality control of true strains of this bacterium. Lipids of taxonomic value (fatty and mycolic acids) are similar in 'M. habana' and M. simiae; nevertheless, they clearly differ on the basis of glycopeptidolipid (GPL) composition. Thus, contrary to M. simiae, most strains of 'M. habana' can be defined by the presence of three polar compounds, designated GPL-I, GPL-II and GPL-III, easily determined by thin-layer chromatography, and characterized, respectively, by the content of l-fucose, 2,4-di-O-Me-d-glucuronic acid, and 4-O-Me-d-glucuronic acid, as epitopes.

Animals↗

Nontypeable Haemophilus influenzae: challenges in developing a vaccine.

Nontypeable Haemophilus influenzae (NTHi) is a gram-negative coccobacillus that is one of the bacteria that form the commensal flora of the upper respiratory tract in humans. This bacterium is an important human pathogen causing a broad spectrum of disease in both adults and children, including invasive and localised infections. The challenges in developing a bacterial protein antigen into an effective vaccine are, firstly, understanding what factors constitute an effective protective immune response for the host, and secondly, to design an effective delivery system that can target and induce the required immune response in humans that will prevent the variety of infections caused by NTHi.

Adult↗

Intranasal immunization with Chlamydia trachomatis, serovar E, protects from a subsequent vaginal challenge with the homologous serovar.

To vaccinate against a vaginal challenge with Chlamydia trachomatis, C3H/HeJ (H-2k) mice were immunized intranasally (i.n.) or intraperitoneally (i.p.) with 1 x 10(6) inclusion forming units (IFU) of C. trachomatis, serovar E and i.n. with 1 x 10(6) UV inactivated IFU of serovar E. Animals inoculated i.n. with mock infected HeLa 229 cells were used as controls. Upon a vaginal challenge with 5 x 10(3) IFU of serovar E, mice immunized i.n. with viable serovar E exhibited significant protection as judged by the number of mice infected compared to controls (p < 0.05). In contrast, mice immunized i.n. with serovar E that had been UV-inactivated, were not protected from a subsequent vaginal challenge with serovar E. Mice immunized i.p. with serovar E showed attenuation of the infection by 4 weeks after challenge compared to control mice as to the number of animals with positive vaginal cultures (p < 0.05). Of the immune parameters examined, the best correlation with protection was seen with Chlamydia specific IgG and IgA vaginal titers and lymphoproliferative responses to serovar E. In summary, mucosal immunization with viable serovar E partially protected mice against a subsequent vaginal challenge, thereby showing that it is possible to elicit a protective response to a human strain of C. trachomatis at a distant mucosal site in this animal model.

Administration, Intranasal↗

Two distinct ospA genes among Borrelia valaisiana strains.

Borrelia valaisiana is a recently described bacterial species in the Borrelia burgdorferi sensu lato complex. To further characterize this bacterium, the plasmid-encoded ospA genes from eight B. valaisiana isolates were amplified by PCR, cloned and sequenced. All B. valaisiana isolates studied possessed an ospA gene with a size of 822-825 bp. The identity of the predicted amino acid sequences of the OspA proteins among B. valaisiana isolates was 69.1-100%, and ranged from 68.2 to 79.1% between B. valaisiana and other B. burgdorferi sensu lato species. Based on the OspA protein sequences, the eight B. valaisiana isolates could be distinguished into two subgroups. Subgroup I contained six B. valaisiana isolates of which OspA sequences were almost identical, but clearly differed from other LB spirochetes. Subgroup II consisted of two isolates with identical OspA sequences which were only 70% identical to subgroup I B. valaisiana isolates and similarly distant from the OspA sequences of other B. burgdorferi sensu lato genospecies. Phylogenetic analysis indicates that B. valaisiana isolates belonging to subgroups I and II possibly evolved from two distinct ancestors. Our data showed for the first time a major difference in OspA proteins within a well-defined B. burgdorferi sensu lato species at the evolutionary level, suggesting that it is not always reliable to assign Borrelia isolates to a definite species solely based on data from ospA gene sequence analysis.

Amino Acid Sequence↗

Use of ribotyping, IS200 typing and plasmid analysis for the identification of Salmonella enterica subsp. enterica serovar Typhimurium vaccine strain Zoosaloral H and its differentiation from wild type strains of the same serovar.

Fifty Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) isolates obtained from one vaccinated and three non-vaccinated poultry flocks as well as the commercially available vaccine strain Zoosaloral H and a S. Typhimurium reference strain were characterized genotypically to differentiate between S. Typhimurium live vaccine strain Zoosaloral H and wild type strains of the same serovar. Ribotyping revealed five different patterns one of which exclusively occurred in the vaccine strain. Seven different hybridization patterns could be observed by IS200-typing of the S. Typhimurium isolates; one of them was only detectable in the vaccine strain. Plasmid analysis showed that 51% of the S. Typhimurium isolates including the vaccine strain harboured large plasmids of approximately 60 MDa. Hybridization with a virulence gene probe identified only 48% of these large plasmids, including that of the vaccine strain, to carry this virulence-associated gene. However, restriction endonuclease analysis of the hybridizing plasmids showed that the virulence gene was located on HindIII fragments of different sizes in the plasmid of the S. Typhimurium vaccine strain Zoosaloral H and in the plasmids of the respective wild type S. Typhimurium isolates. Thus, ribotyping, IS200-typing and plasmid analysis represent at least three independent systems which allow the genotypic identification of the S. Typhimurium vaccine strain Zoosaloral H and its differentiation from wild type isolates of the same serovar.

Animals↗

Antigenic variation in Porphyromonas gingivalis ribotypes recognized by serum immunoglobulin G of adult periodontitis patients.

We obtained clinical isolates of Porphyromonas gingivalis of known ribotype from patients diagnosed with adult periodontitis and used Western blot methodology to evaluate profiles of antigens recognized by IgG in heterologous and homologous patient sera. Our aims were to identify isolates belonging to different serogroups, to learn if serogroup membership is related to ribotype to assess variation in IgG responses of patients to antigens is homologous and heterologous ribotypes, and to determine the frequency of shared and variable antigens in different biochemical classes recognized across different serogroups and ribotypes. Blots of separation patterns of 28 isolates were developed in sera from patients and bound IgG was quantified by digital image densitometry. The membership of isolates in different serogroups was determined by correlation and hierarchical cluster analysis of isolate whole-cell IgG binding profiles. Two major isolate clusters, each with two subclusters, were found. Isolates within the same ribotype clustered together in some cases but not others. Homologous isolates ranked high in IgG binding levels relative to those from different patients irrespective of ribotype. Patient subgroups with IgG responses dominant for different ribotypes and serogroups were revealed by correlation analysis. The IgG binding profiles observed for individual protein and proteinase-resistant antigens across both homologous and heterologous isolates were very dissimilar. Furthermore, the frequency of antigens both shared across all ribotypes and recognized by IgG in patient sera was unexpectedly low. Only two protein antigens (Mr 44 kDa and 27 kDa) were strongly recognized across all ribotypes by different sera. We conclude that the IgG response of patients infected with a particular P. gingivalis serotype or ribotype is directed mainly against antigens that are not shared by other potentially infective clonal types.

Adult↗

High diversity of group A Streptococcal emm types in an Indian community: the need to tailor multivalent vaccines.

BACKGROUND: Concern about the emergence of antibiotic-resistant strains and about morbidity and/or mortality related to rheumatic fever and rheumatic heart disease has been a continuous impetus for the development of a safe, effective vaccine against group A Streptococcus (GAS). To date, >120 GAS M types are known, as identified by serological typing. In general, serum immunoglobulin G directed to the hypervariable NH2 terminal portion of M protein leads to complement fixation and opsonophagocytosis of the homologous streptococcal serotype by polymorphonuclear leukocytes, and the protection is type specific. The sequence variation at the N terminus ultimately affects the binding of opsonic antibodies. Because of hypervariability in these opsonic sequences from different M types, it was relevant to use epitopes derived from these multiple sequences in a "multivalent vaccine" design for evaluation of protection against these M types of GAS. Thus, any attempts to design vaccines for a given community will require information on N terminal-sequence typing and variation. METHODS: In the present study, we performed molecular characterization of isolates recovered from patients in northern India--to our knowledge, for the first time--in an attempt to study the circulating M types and their N terminal sequence variability. RESULTS: We report tremendous diversity in GAS strains recovered from symptomatic patients, with implications on the design of appropriate vaccines. Fifty-nine isolates represented 33 different sequence types. Very few novel types and no predominant clones were found. CONCLUSIONS: The high diversity of emm types encountered in a single year suggests that any M protein-based multivalent vaccine would have to be specifically tailored for this region.

Bacterial Typing Techniques↗

Changes in the distribution of Haemophilus influenzae type b clones associated with widespread infant vaccination in Finland.

Isolates from 646 consecutive Finnish Haemophilus influenzae type b (Hib) patients with systemic disease, collected before and during large-scale vaccinations with Hib conjugate vaccines, were analyzed by major outer membrane protein (OMP) subtyping, lipopolysaccharide (LPS) serotyping, and biotyping (BT). Strains with OMP-BT-LPS combinations (clones) 1-I-1 and 1c-I-1 disappeared at the same rate as the disease they were associated with. A preferential decrease in the number of isolates of clone 1-II-1 was recorded, whereas the reduction in disease caused by strains of clone 1-II-9 occurred at a lower rate than expected. The latter clone occurred mainly in the most densely populated area of Finland. Strains belonging to all the common Hib clones were isolated from the 16 infants who acquired Hib disease despite being (partially) vaccinated. Thus, Hib clones disappeared during mass vaccination with conjugate vaccines, although at different rates.

Adolescent↗

Antigenic analysis of iron-regulated proteins in Pasteurella haemolytica A and T biotypes by immunoblotting reveals biotype-specific epitopes.

The antigenic relationships of the iron-regulated proteins (IRPs) in Pasteurella haemolytica A and T biotype strains were examined by SDS-PAGE and immunoblotting. P. haemolytica cells of the A biotype, grown under conditions of iron-limitation, expressed two IRPs, of 35 and 70 kDa. All T biotype strains expressed IRPs with slightly different molecular masses of 37 and 78 kDa. Immunoblotting of all 16 P. haemolytica serotypes was carried out using a panel of polyclonal and monoclonal antibodies raised against serotype A2 antigens. Polyclonal antibodies revealed inter-serotype cross-reactivity towards the 35 and 70 kDa IRPs within the A biotype but no cross-reactivity against a T biotype protein in the 78 kDa region. Monoclonal antibody against the 35 kDa antigen reacted only with the A biotype 35 kDa IRP. Identical profiles were obtained for 10 field isolates of serotype A2, further emphasizing the antigen conservation within the A biotype. These findings reinforce the view that the A and T biotypes of P. haemolytica should be considered as separate species and suggest that IRPs from single A and T biotype strains incorporated into a vaccine might provide cross-protection against all P. haemolytica serotypable strains. Similar studies on the IRPs of 10 untypable strains revealed some of these to have different antigenic reactivities from those observed within the A and T biotypes.

Antibodies, Bacterial↗

Molecular characterization of Salmonella enterica subsp. enterica serovar Typhimurium DT009 isolates: differentiation of the live vaccine strain Zoosaloral from field isolates.

A total of 25 epidemiologically unrelated Salmonella enterica subsp. enterica (S.) serovar Typhimurium DT009 isolates from various human and animal sources, the original S. Typhimurium DT009 Zoosaloral live vaccine strain and two Zoosaloral strains reisolated from vaccinated chickens were investigated by various molecular typing methods (I) to determine the most suitable method or combination of methods for the differentiation of DT009 field isolates and (II) to investigate which molecular methods are suitable to differentiate the Zoosaloral live vaccine strain from field isolates of the same phage type. Based on the results of plasmid profile analysis, IS200 typing and macrorestriction analysis with XbaI, SpeI and BlnI, the 28 S. Typhimurium DT009 isolates were assigned to 16 different genomic groups, one of which was exclusively represented by the original and the reisolated Zoosaloral strains. IS200 typing was the most discriminatory single method for the differentiation of the DT009 isolates followed by plasmid profile analysis and BlnI-macrorestriction analysis. The Zoosaloral vaccine strain differed from the DT009 field isolates by its unique HindIII-fragment pattern of the virulence plasmid and by its unique SpeI-macrorestriction pattern.

Animals↗