Carriage of Neisseria meningitidis Among Household Contacts of Patients with Meningococcal Disease in New Zealand

 The aims of this study were to estimate carriage prevalence, identify factors predictive of carriage, and compare strains of Neisseria meningitidis isolated from patients with meningococcal disease and their household contacts. A total of 954 contacts of 160 patients had a nasopharyngeal swab and an interview relating to factors associated with carriage. The carriage prevalence was 20.4% for Neisseria meningitidis, 11.3% for serogroup B, and 2.6% for serogroup C. Age-standardised carriage was higher in Maori (36.8%) than in Pacific Island (21.5%) or European/other (11.1%) ethnic groups. Factors associated with carriage were smoking, with personal smokers (odds ratio [OR] 2.5) and passive smokers (OR 1.6) having a higher carriage risk than those in smoke-free houses; ethnicity, with Maoris having a higher carriage risk than those of non-Maori or non-Pacific Island ethnicity (OR 2.2); gender, with males at higher risk than females (OR 1.7); and age, with 0–4-year-olds less likely and 15–24-year-olds more likely to be carriers than those over 25 years. Strong patient-contact clustering by meningococcal strain (chi-square₁=16.7, P=0.00004) suggested an important role for the household setting in transmission. The low carriage prevalence of serogroup B Neisseria meningitidis among household contacts may reflect its low transmissibility but high virulence. No direct relationship was found between prevalence of ethnic-specific carriage and the incidence of meningococcal disease.     

Characterization of Neisseria meningitidis Strains Causing Disease in Complement-Deficient and Complement-Sufficient Patients

Serotyping and serosubtyping of meningococci showed no difference between isolates from 44 complement-deficient persons and from 50 complement-sufficient persons with meningococcal disease. Multilocus enzyme electrophoretic typing of the meningococci revealed 54 electrophoretic types that were equally distributed among isolates from complement-deficient and complement-sufficient patients. Analysis of strains isolated from eight complement-deficient persons with 11 recurrences of meningococcal disease showed that one strain was identical to the strain previously isolated from the same individual. Our results indicate that there are no differences between the clonal distributions of strains infecting complement-deficient and complement-sufficient patients. Most recurrences were infections caused by different strains.     

Molecular and Serological Diversity of Neisseria meningitidis Carrier Strains Isolated from Italian Students Aged 14 to 22 Years

Neisseria meningitidis is an obligate human commensal that commonly colonizes the oropharyngeal mucosa. Carriage is age dependent and very common in young adults. The relationships between carriage and invasive disease are not completely understood. In this work, we performed a longitudinal carrier study in adolescents and young adults (173 subjects). Overall, 32 subjects (18.5%) had results that were positive for meningococcal carriage in at least one visit (average monthly carriage rate, 12.1%). Only five subjects tested positive at all four visits. All meningococcal isolates were characterized by molecular and serological techniques. Multilocus sequence typing, PorA typing, and sequencing of the 4CMenB vaccine antigens were used to assess strain diversity. The majority of positive subjects were colonized by capsule null (34.4%) and capsular group B strains (28.1%), accounting for 23.5% and 29.4% of the total number of isolates, respectively. The fHbp and nhba genes were present in all isolates, while the nadA gene was present in 5% of the isolates. The genetic variability of the 4CMenB vaccine antigens in this collection was relatively high compared with that of other disease-causing strain panels. Indications about the persistence of the carriage state were limited to the time span of the study. All strains isolated from the same subject were identical or cumulated minor changes over time. The expression levels and antigenicities of the 4CMenB vaccine antigens in each strain were analyzed by the meningococcal antigen typing system (MATS), which revealed that expression can change over time in the same individual. Future analysis of antigen variability and expression in carrier strains after the introduction of the MenB vaccine will allow for a definition of its impact on nasopharyngeal/oropharyngeal carriage.     

Phenotypical and Genotypical Characterization of Neisseria meningitidis Carrier Strains Isolated from Polish Recruits in 1998

 The aim of this study was to investigate the Neisseria meningitidis carriage rate among two cohorts of Polish recruits upon entry to the military and during the first 2 months of their service, i.e. in the spring and autumn of 1998, and to characterize the meningococcal strains isolated. Pharyngeal swabs were taken four and five times from 151 and 168 men, respectively. Altogether, 81 and 180 meningococcal isolates representing 54 and 102 different strains were recovered. The overall rates of carriage in the spring and in the autumn were 36% and 61%, and, among recruits who submitted to sampling on at least three occasions, 39% and 55%. Eighty-three of 156 (53%) meningococcal carrier strains were nongroupable; among the remaining strains, serogroup B was predominant (32% of all carrier strains). In both surveys the predominant phenotype was Neisseria meningitidis NG : 21 : P1.7.     

Immunological and Molecular Characterization of Three Variant Subtype P1.14 Strains of Neisseria meningitidis

Epidemic outbreaks of group B meningococcal disease exhibit a clonal nature consisting of a common serotype-subtype. Subtype-specific monoclonal antibodies (MAbs) directed toward two variable regions (VR1 and VR2) of the class 1 protein of Neisseria meningitidis are used in this classification scheme. A new MAb was developed to classify a nonsubtypeable (NST) strain of N. meningitidis, 7967. This MAb bound to both the NST strain and the prototype subtype P1.14 strain, S3446, by dot blot analysis. However, a MAb produced to the prototype P1.14 strain did not bind to strain 7967. Sixteen additional strains were further identified as P1.14 with the prototype MAb; of these, 15 strains bound both MAbs. Differences in the characteristics of binding of both antibodies to the three apparently diverse P1.14 strains were studied further by using outer membrane complex proteins, immobilized peptides, and soluble peptides. Deduced amino acid analysis suggested that both MAbs bind to VR2 and that single amino acid changes within VR2 (KM, NM, or KK) might explain the differences in binding characteristics. These results demonstrated that minor variations which exist within subtype variable regions may be clearly identified only by a combination of molecular and immunologic testing. The impact of subtype variation will become more evident as subtype-specific vaccines are developed and tested for efficacy.Neisseria meningitidis causes bacterial meningitis and septicemia worldwide. Epidemiological studies of endemic or epidemic outbreaks of meningococcal disease rely on polyvalent and monoclonal antibodies (MAbs) to capsular polysaccharides for monitoring serogroup specificity (27). This organism can be further classified into serotypes and subtypes with MAbs directed toward two major outer membrane proteins (OMPs) (6). Serotyping is based on MAbs which bind to the class 2 or class 3 OMP (PorB). Subtyping MAbs bind one of two immunodominant variable regions (VR1 and VR2) of the class 1 OMP (PorA) (1, 9, 10, 11, 13, 22). Complete subtyping requires identification of epitopes found in both variable regions (5).MAbs to about 15 subtype epitopes have been developed and characterized; however, nonsubtypeable (NST) strains are still found. NST strains represent one of three categories: (i) strains possessing class 1 epitopes to which MAbs have not been developed and characterized, (ii) strains which do not express PorA, and (iii) strains in which the PorA subtype epitopes differ only slightly from known subtype epitopes due to genetic modifications, such as point mutations or duplication or deletion events, which eliminate binding of subtyping MAbs.A significant part of recent group B meningococcal vaccine development efforts has been focused on OMPs as principal components of a subtype-serotype-specific vaccine (26). This vaccination approach is based on the observations that PorA elicits a human bactericidal antibody response (24) and that subtype-specific MAbs passively protect infant rats against challenge with N. meningitidis (17, 18). An effective subtype-specific vaccine should include the most prevalent subtype epitopes associated with the strains causing disease in the population in which the vaccine will be used. Human bactericidal antibodies induced by vaccination with a vaccine of one subtype are not equally effective in killing other subtype strains. Even single amino acid changes in VR1 and VR2 and deletions in regions flanking the epitopes may result in loss of reactivity with subtype-specific MAbs (12, 23), as observed in several recent outbreaks. One such variant also showed increased resistance to bactericidal activity (16), suggesting a possible influence of such subtype variants on the level of protection induced by a subtype-specific vaccine.We have defined three different point mutations in the subtype-specific epitope P1.14 of N. meningitidis, using a combination of molecular and immunological techniques. The variability within this epitope highlights the importance of defining subtypes molecularly as well as immunologically.     

Molecular Characterization of Methicillin-Resistant Staphylococcus aureus Strains Isolated from Patients with Hospital Readmissions

It is unclear whether patients colonized with methicillin-resistant Staphylococcus aureus (MRSA) continue to harbor the same genotype during hospital readmissions. We characterized 140 MRSA strains isolated from 33 persistent MRSA carriers with hospital readmissions. Nearly half of the patients continued to harbor the same genotype, and the rest acquired different genotypes. Among 25 patients who received eradication therapy, 16 (64%) were colonized with MRSA strains exhibiting different genotypes from the preexisting one.     

Strains of Neisseria meningitidis isolated from patients and their close contacts.

Neisseria meningitidis isolates from contacts, mostly family members, of 27 unrelated meningococcal disease patients were examined by serogrouping, serotyping, and a recently described sodium dodecyl sulfate-polyacrylamide gel electrophoresis typing procedure. Most of the isolates were serogroup B or C. Serotyping and sodium dodecyl sulfate-polyacrylamide gel electrophoresis typing now provide a more precise means than serogrouping for determining the epidemiological relationships among patient isolates and those of related carriers. In 70% of the families studied, all contact carriers had strains indistinguishable from that of the patient. In the other 30%, more than one meningococcal strain was recovered from the family. Sixty percent of the carrier isolates were recovered from adults. It was found that, among household contacts, the mother was most likely and the father was least likely to carry the disease isolate. Nonhousehold contacts were least likely to carry the disease isolate.     

Genetic Study of Capsular Switching between Neisseria meningitidis Sequence Type 7 Serogroup A and C Strains

Neisseria meningitidis is a leading cause of septicemia and meningitis worldwide. N. meningitidis capsular polysaccharides have been classified into 13 distinct serogroups which are defined by antibody reactivity and structural analysis, and the capsule plays an important role in virulence. Serogroups A, B, C, W135, and Y have been reported to be clinically important. Several newly identified serogroup C isolates belonging to the unique sequence type 7 (ST-7) were identified in China. Since most ST-7 isolates from China belonged to serogroup A, the newly identified ST-7 serogroup C strains were proposed to have arisen from those belonging to ST-7 serogroup A. In this study, six ST-7 serogroup C and three ST-7 serogroup A isolates were analyzed by pulsed-field gel electrophoresis to confirm their sequence type. In order to clarify the genetic basis of capsular switching between ST-7 serogroup A and C strains, the whole capsular gene clusters and surrounding genes of the two representative ST-7 strains belonging to serogroups A and C, respectively, were sequenced and compared. Potential recombination sites were analyzed using the RDP3 beta software, and recombination-related regions in two other ST-7 serogroup A and five ST-7 serogroup C strains were also sequenced and compared to the representative ST-7 serogroup A and C strain sequences.Neisseria meningitidis is a leading cause of septicemia and meningitis (13), and the extracellular polysaccharide capsule is a prerequisite for meningococcal virulence (19). As a consequence of capsular polysaccharide structural differences, 13 N. meningitidis serogroups have been identified, and the isolates most frequently associated with human disease belong to serogroups A, B, C, W135, and Y (9, 14). Serogroups B and C are responsible for most human infections in Europe and America, and serogroup A and C infections are more common in Africa and Asia (11). Antigenic diversity between N. meningitidis strains resulting from DNA transformation and/or subsequent recombination of capsular genes has been described (2, 15). Although the genetic mechanisms involved in the switching of capsular genes from B to C followed by switching from C to W135 have been described, the recombination sites associated with these events have not been clearly defined (2, 15).In China, the predominant genotype of serogroup C N. meningitidis (menC) is sequence type 4821 (ST-4821), accounting for about 75% of menC found in 12 provinces (10). Previously, we identified several isolates in China belonging to the unique ST-7 and serogroup C by using multilocus sequence typing (MLST), porA typing, and comparative genomic hybridization (CGH) analyses (10). Since the majority of ST-7 isolates in China belong to serogroup A, we hypothesized that the newly identified ST-7 serogroup C arose from ST-7 serogroup A strains.In this report, we analyze several ST-7 serogroup A and C isolates by comparing their respective whole capsular gene clusters and their surrounding regions. This analysis describes the genetic basis of the capsular switching events that resulted in the establishment of ST-7 serogroup C strains and identifies the recombination sites involved in the genetic exchange which gave rise to this novel serogroup.     

Clonal Distribution of Disease-Associated and Healthy Carrier Isolates of Neisseria meningitidis between 1983 and 2005 in Cuba

In response to epidemic levels of serogroup B meningococcal disease in Cuba during the 1980s, the VA-MENGOC-BC vaccine was developed and introduced into the National Infant Immunization Program in 1991. Since then the incidence of meningococcal disease in Cuba has returned to the low levels recorded before the epidemic. A total of 420 Neisseria meningitidis strains collected between 1983 and 2005 in Cuba were analyzed by multilocus sequence typing (MLST). The set of strains comprised 167 isolated from disease cases and 253 obtained from healthy carriers. By MLST analysis, 63 sequence types (STs) were identified, and 32 of these were reported to be a new ST. The Cuban isolates were associated with 12 clonal complexes; and the most common were ST-32 (246 isolates), ST-53 (86 isolates), and ST-41/44 (36 isolates). This study also showed that the application of VA-MENGOC-BC, the Cuban serogroup B and C vaccine, reduced the frequency and diversity of hypervirulent clonal complexes ST-32 (vaccine serogroup B type-strain) and ST-41/44 and also affected other lineages. Lineages ST-8 and ST-11 were no longer found during the postvaccination period. The vaccine also affected the genetic composition of the carrier-associated meningococcal isolates. The number of carrier isolates belonging to hypervirulent lineages decreased significantly after vaccination, and ST-53, a sequence type common in carriers, became the predominant ST.Neisseria meningitidis is a major cause of meningitis and septicemia worldwide, and meningococcal disease (MD) remains one of the most aggressive bacterial infectious diseases in humans. Only 5 of the 13 distinct N. meningitidis serogroups, which are determined on the basis of the immunochemistry of their capsular polysaccharides, are associated with invasive disease; of these, serogroups A, B, and C account for approximately 90% of all cases (42). The disease mainly affects infants and young children, with case fatality rates being 10% to 15%, despite the availability of effective antibiotics. Although early diagnosis and antibiotic treatment greatly enhance survival, prevention through vaccination constitutes the best way to control meningococcal disease (21).Traditionally, isolates of N. meningitidis have been identified and characterized phenotypically on the basis of the recognition of meningococcal surface structures by specific monoclonal or polyclonal antibodies (2, 19). While serogrouping, serotyping, and serosubtyping, based on the detection of variants of capsular polysaccharide, PorB and PorA, respectively, are still used extensively, they have often been found to lack resolution, especially when they are used to work with endemic strains (11, 51).The polysaccharide capsule is the major virulence factor of N. meningitidis; unencapsulated meningococci are essentially avirulent (52). The development of comprehensive capsule-based vaccines has, however, been constrained by the poor immunogenicity of the serogroup B capsule (53) and by concerns over the antigenic similarity of this molecule with host sialic acids. Although the focus of vaccine development efforts for serogroup B has consequently been switched toward the meningococcal outer membrane proteins, the large degree of antigenic variation exhibited by these antigens as a result of the selection pressure imposed by the immune system has become the most important challenge to both serogroup B vaccine development and epidemiological studies. Understanding this variation in terms of the genetic structure of meningococcal populations is, therefore, of paramount importance for steering vaccine research and analyzing the present and future performance of existing vaccination approaches.Meningococcal populations are known to be genetically highly diverse. Many genotypes have been identified by the examination of housekeeping genes that are subject to selection for conserved metabolic function (8, 24, 28, 55). These genotypes have been identified as electrophoretic types by multilocus enzyme electrophoresis (MLEE) (44) and, more recently, as sequence types (STs) by multilocus sequence typing (MLST) (28). Population studies that exploit MLEE and MLST have identified equivalent groups of related genotypes that are referred to as clonal complexes (49); which are now named after a predominant, or central, ST. Isolate collections corresponding to populations of asymptomatically carried meningococci show the greatest genetic diversity, while most disease-associated meningococci belong to a limited number of clonal complexes known as hyperinvasive lineages (5, 7, 13, 24, 55).MD in Cuba has not been considered a public health problem since the introduction of an outer membrane vesicle (OMV)-based vaccine (VA-MENGOC-BC) in the 1980s (16, 41, 46). The incidence of MD in the country slowly increased from a reported figure of 0.1 cases per 100,000 inhabitants in 1962 to 1.8 cases per 100,000 inhabitants in 1978, with most cases caused by serogroup C isolates. In spite of nationwide vaccination in 1979 with a capsular polysaccharide A-C vaccine (Merieux Laboratories), the incidence rate continued to rise and was accompanied by a shift to predominantly serogroup B among disease-associated isolates. The peak of the epidemic occurred during 1983 and 1984, reaching a maximum incidence of 14.4 cases per 100,000 inhabitants; most cases at this point were caused by a single serogroup B clone. In the 1980s, research started in Cuba on an OMV-based serogroup B-C vaccine developed from the outer membrane antigens of the epidemic type strain (strain CU385) combined with serogroup C polysaccharide. The resulting formulation (VA-MENGOC-BC) was shown to be safe and effective, and in 1991 the vaccine was included in the National Immunization Program (NIP) for all infants (45, 46). After its introduction in the vaccination campaign in 1989 (in which the population between 3 months and 24 years old was vaccinated), the overall incidence decreased to below 0.4 per 100,000 inhabitants by 2001 and to 0.1 cases per 100,000 inhabitants by 2008 (34).Epidemiological studies conducted in Cuba have used typing methods based on phenotypic traits (30, 31), which lack discriminatory power for the study of the genetic structure of bacterial populations or for monitoring of the changes during outbreaks and epidemics. The present study, therefore, was aimed at obtaining further information about the Cuban epidemic on the basis of the characterization by MLST of N. meningitidis strain isolates collected on the island during a 22-year period (1983 to 2005) and to use this information to begin trying to understand the impact of mass vaccination with the Cuban VA-MENGOC-BC vaccine on the genetic structure of the N. meningitidis population isolated from cases and carriers.(Part of this study was presented at the 16th International Pathogenic Neisseria Conference, Rotterdam, The Netherlands, September 2008.)     

Homology of cryptic plasmid of Neisseria gonorrhoeae with plasmids from Neisseria meningitidis and Neisseria lactamica.

DNA probe hybridisation was used to examine the relation between the cryptic plasmid from Neisseria gonorrhoeae and plasmids carried by pharyngeal isolates of Neisseria meningitidis and Neisseria lactamica. The complete gonococcal cryptic plasmid and HinfI derived digestion fragments subcloned into Escherichia coli were used to probe Southern blots of plasmid extracts. Homology was found to a plasmid of approximate molecular weight 4.5 kilobase pairs (Kb) but not to plasmids of less than 3.2 Kb or 6.5 Kb. Eleven of 16 strains of N meningitidis and two of six strains of N lactamica carried plasmids that showed strong hybridisation with the 4.2 Kb gonococcal plasmid. Hybridisation of plasmids from non-gonococcal species of neisseria with the gonococcal cryptic plasmid indicates that caution should be taken when using the cryptic plasmid as a diagnostic probe for gonorrhoea.     

Protein Fraction with Immunogenic Potential and Low Toxicity Isolated from the Cell Wall of Neisseria meningitidis Group B

Several fractions were extracted from the cell envelope (CE) of Neisseria meningitidis group B and characterized with regard to their morphology, antigenicity, protein composition, and toxicity. Whole bacterial cells were suspended in a medium of low ionic strength and disrupted in a French pressure cell. The crude CE thus obtained were separated into cell membrane (CM) enriched and cell wall (CW) enriched fractions on sucrose density gradients. In addition, CM and CW fractions were separated from CE on the basis of differential solubility in the nonionic detergent, Triton X-100. The Triton-insoluble fraction, containing primarily CW components, was further treated with a mixture of Triton and ethylenediaminetetraacetic acid, which was shown to remove additional protein and most of the lipopolysaccharide. Electron microscope examination of the various fractions revealed typical unit membrane structures in the case of CM, or large, open segments in the case of CW. The Triton-insoluble and especially the Triton-ethylenediaminetetraacetic acid-insoluble fractions consisted of small vesicular structures. All fractions, except the Triton-soluble fraction, when assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were shown to contain one major protein component accounting for more than 50% of the total. Sera from rabbits immunized with the various fractions formed precipitin lines in immunodiffusion tests against the homologous and some of the heterologous fractions. High-titer bactericidal antibodies were also demonstrated in these sera when tested against the homologous strains. Toxicity studies in rats sensitized with lead acetate indicate that the level of contamination of Triton-insoluble/Triton-ethylenediaminetetraacetic acid-insoluble fractions with lipopolysaccharide was significantly smaller than that of the other fractions.     

Cytotoxic Effects of Klebsiella oxytoca Strains Isolated from Patients with Antibiotic-Associated Hemorrhagic Colitis or Other Diseases Caused by Infections and from Healthy Subjects

Antibiotic-associated hemorrhagic colitis (AAHC) is associated with Klebsiella oxytoca. This study analyzed whether cytotoxic properties are linked to specific subtypes of K. oxytoca. Klebsiella isolates from stools of AAHC patients, healthy carriers, and diarrhea patients as well as from infections of other organs were investigated. Cytotoxic effects on human epithelial cells were limited to the species K. oxytoca and were not detectable for any other Klebsiella species. Isolates from AAHC patients and from stools showed the highest proportion of cytotoxic strains. Urinary or respiratory tract isolates exhibited no cytotoxicity. Macrorestriction profiling of strains revealed no genetic relationships of AAHC isolates or the cytotoxic phenotype but identified that different K. oxytoca strains with different cytotoxic behaviors may be prevalent in the same AAHC patient. Under laboratory conditions, cytotoxicity was maximally effective after exponential bacterial growth and then declined despite the continued viability of K. oxytoca cells in culture. Given its capacity to induce AAHC and that a high proportion of stool isolates tested cytotoxin positive, we argue that K. oxytoca should be considered an opportunistic pathogen if detected in stools. The ability to induce disease after antibiotic treatment most likely represents an overgrowth of the toxin-producing bacterium due to an alteration of the normal colonic microflora.Antibiotic-associated colitis (AAC) is a frequent adverse effect observed when the normal bacterial flora is altered due to antibiotic therapy. Most cases of AAC are caused by infection by and extensive growth of Clostridium difficile, leading to pseudomembranous colitis. A special form of AAC is antibiotic-associated hemorrhagic colitis (AAHC), which was first described in 1978 (21) and has since been ascribed specific clinical, endoscopic, histopathological, and microbiological characteristics (9, 10). AAHC is not associated with C. difficile and was only recently shown to be caused by Klebsiella oxytoca (9, 10). AAHC is typically observed after a brief therapy with penicillins, with a sudden onset of bloody diarrhea often in combination with severe abdominal cramps, which often requires hospitalization. The key features of AAHC upon endoscopy are mucosal hemorrhage and mucosal edema, usually with segmental distribution, commonly affecting the ascending colon and the cecum (9). Histology typically resembles that of colitis induced by toxin-producing bacteria (10).For the majority of patients with AAHC, stool testing reveals K. oxytoca in significant amounts (>10⁶ CFU/ml) (9, 23). This Gram-negative rod is ubiquitous in the environment (e.g., soil and water) but can also be isolated from skin, mucous membranes, and the intestines of humans and animals (19). Human infections with K. oxytoca resemble those with Klebsiella pneumoniae; i.e., respiratory and urinary tracts are commonly affected (e.g., nosocomial pneumonia), in addition to soft tissue and hepatobiliary infections (6). Until recently, K. oxytoca was not considered to be an intestinal pathogen, and its presence in stool has placed the organism as a constituent of the normal gut microflora. For the healthy population, colonization of the intestine with K. oxytoca has been reported for 1.6% to 9% of subjects (1, 4, 10, 23). It is important that K. oxytoca constitutively produces β-lactamases conferring resistance to amino- and carboxypenicillins (13), agents typically given before the onset of AAHC. The association of K. oxytoca with AAHC was previously established by an animal model using a K. oxytoca strain isolated from a patient with AAHC in combination with an antibiotic to induce right-sided hemorrhagic colitis (10).In the 1990s, two independent groups reported that K. oxytoca strains isolated from patients with AAHC produce a cytotoxin, which caused cell death in cultured Hep2, Vero, CHO-K1, and HeLa cell lines as well as in an isolated intestinal-loop model (8, 15-17). In contrast, two laboratory strains of K. oxytoca exhibited no cytotoxicity, indicating that cytotoxin production might be strain specific (16, 17). The cytotoxin was reported to be heat labile, insensitive to proteinase digestion, and of a low molecular mass (8, 16, 17). A detailed analysis of the chemical nature and molecular structure of the cytotoxin is not yet available. Moreover, a causal link between toxin production in K. oxytoca and AAHC has not been established.The aim of the current study was to assess whether cytotoxin production is specific to certain subtypes of K. oxytoca and to test the hypothesis that AAHC uniquely correlates with those strains. A total of 121 Klebsiella isolates were investigated, including K. oxytoca strains isolated from stool samples from patients with AAHC, healthy carriers, and patients with colitis/diarrhea of other causes. K. oxytoca strains from infections involving other body sites and other Klebsiella spp. were analyzed in comparison. The characterization of all isolates was performed by using genotypic and biochemical methods, and the capacity of each isolate to induce cytotoxic effects on cultured eukaryotic cells was measured. A subset of strains was genotyped by macrorestriction profiling to assess their genetic relatedness.     

Molecular and biological analysis of eight genetic islands that distinguish Neisseria meningitidis from the closely related pathogen Neisseria gonorrhoeae

The pathogenic species Neisseria meningitidis and Neisseria gonorrhoeae cause dramatically different diseases despite strong relatedness at the genetic and biochemical levels. N. meningitidis can cross the blood-brain barrier to cause meningitis and has a propensity for toxic septicemia unlike N. gonorrhoeae. We previously used subtractive hybridization to identify DNA sequences which might encode functions specific to bacteremia and invasion of the meninges because they are specific to N. meningitidis and absent from N. gonorrhoeae. In this report we show that these sequences mark eight genetic islands that range in size from 1.8 to 40 kb and whose chromosomal location is constant. Five of these genetic islands were conserved within a representative set of strains and/or carried genes with homologies to known virulence factors in other species. These were deleted, and the mutants were tested for correlates of virulence in vitro and in vivo. This strategy identified one island, region 8, which is needed to induce bacteremia in an infant rat model of meningococcal infection. Region 8 encodes a putative siderophore receptor and a disulfide oxidoreductase. None of the deleted mutants was modified in its resistance to the bactericidal effect of serum. Neither were the mutant strains altered in their ability to interact with endothelial cells, suggesting that such interactions are not encoded by large genetic islands in N. meningitidis.     

Whole-Genome Comparison of Aspergillus fumigatus Strains Serially Isolated from Patients with Aspergillosis

The emergence of azole-resistant strains of Aspergillus fumigatus during treatment for aspergillosis occurs by a mutation selection process. Understanding how antifungal resistance mechanisms evolve in the host environment during infection is of great clinical importance and biological interest. Here, we used next-generation sequencing (NGS) to identify mutations that arose during infection by A. fumigatus strains sequentially isolated from two patients, one with invasive pulmonary aspergillosis (IPA) (five isolations) and the other with aspergilloma (three isolations). The serial isolates had identical microsatellite types, but their growth rates and conidia production levels were dissimilar. A whole-genome comparison showed that three of the five isolates from the IPA patient carried a mutation, while 22 mutations, including six nonsynonymous ones, were found among three isolates from the aspergilloma patient. One aspergilloma isolate carried the cyp51A mutation P216L, which is reported to confer azole resistance, and it displayed an MIC indicating resistance to itraconazole. This isolate harbored five other nonsynonymous mutations, some of which were found in the afyap1 and aldA genes. We further identified a large deletion in the aspergilloma isolate in a region containing 11 genes. This finding suggested the possibility that genomic deletions can occur during chronic infection with A. fumigatus. Overall, our results revealed dynamic alterations that occur in the A. fumigatus genome within its host during infection and treatment.     

Determination of Types of Enterocytozoon bieneusi Strains Isolated from Patients with Intestinal Microsporidiosis

To determine the types of Enterocytozoon bieneusi strains associated with intestinal microsporidiosis, we developed a rapid and efficient approach for typing parasites obtained from stool specimens by PCR-restriction fragment length polymorphism (PCR-RFLP). Typing was based on DNA polymorphism of the ribosomal DNA internal transcribed spacer (ITS) region of E. bieneusi. RFLPs generated with two restriction enzymes (NlaIII and Fnu4HI) in PCR-amplified ITS products were used to classify strains into different lineages. This approach was successfully used to differentiate 78 strains that had been obtained from the stools of 65 patients with intestinal microsporidiosis. Among the 78 strains, we found four genetically unrelated lineages, showing the genetic diversity of E. bieneusi. Type I strains of E. bieneusi were found in a majority of the samples, accounting for 51 (78%) of the 65 microsporidiosis cases. In contrast, type II, III, and IV strains were found in only 8 (12%), 3 (5%), and 3 (5%) cases, respectively. All strains of E. bieneusi we have tested so far fall into one of four different lineages, and this study shows that human intestinal microsporidiosis is most often associated with type I strains. PCR-RFLP analysis of the ITS region of E. bieneusi should be useful for epidemiological studies.Microsporidia are obligate intracellular protozoan parasites that can infect vertebrates as well as invertebrates. Over 1,000 species of microsporidia have been described, but reports of human infections were rare before the AIDS epidemic. Since then, microsporidia have been recognized as opportunistic pathogens in patients with AIDS. Different species of microsporidia have been shown to infect humans, and among these species, Enterocytozoon bieneusi (7) is by far the most frequently identified. E. bieneusi infection is responsible for chronic diarrhea and wasting in immunocompromised patients, such as patients with AIDS and organ transplant recipients (2, 15, 17, 23). Substantial variation in the progression and severity of disease is observed among cases of intestinal microsporidiosis, and these differences are presumably due to several factors, including host immune defenses and phenotypic differences in parasite strains (23).However, modes of transmission and sources of human infection by E. bieneusi are largely unknown (11, 23). Epidemiologic investigations of a number of infectious diseases have shown that amplification methods could be extremely valuable tools for laboratory-based investigations (20). This approach has proven successful in defining different strains of Encephalitozoon cuniculi, a zoonotic microsporidian that can infect humans and a wide variety of other mammals (6, 8, 10, 14). At least three strains of this species have been defined, on the basis of the number of 5′-GTTTT-3′ repeats present in the internal transcribed spacer (ITS) region of the ribosomal DNA. We wished to study the genetic diversity of E. bieneusi strains by a similar approach. In this report, we describe the development of an amplification-based assay for genetic analysis of the ITS region of E. bieneusi that should be useful for epidemiological studies.     

Immune Responses against Major Outer Membrane Antigens of Neisseria meningitidis in Vaccinees and Controls Who Contracted Meningococcal Disease during the Norwegian Serogroup B Protection Trial

Sera from vaccinees and controls who contracted serogroup B meningococcal disease during the blinded and open parts of a two-dose protection trial in Norway were compared for antigen-specific and bactericidal antibodies against vaccine strain 44/76 (B:15:P1.7,16). From 16 of 20 (80%) vaccinees and 26 of 35 (74%) controls, one or more serum samples (n = 104) were collected during the acute phase (1 to 4 days), early convalescent phase (5 to 79 days), and late convalescent phase (8 to 31 months) after onset of disease. Binding of immunoglobulin G (IgG) to the major outer membrane antigens (80- and 70-kDa proteins, class 1, 3, and 5 proteins, and lipopolysaccharide [LPS]) on immunoblots was measured by digital image analysis. Specific IgG levels in vaccinees increased from acute to early convalescent phases, followed by a decline, while controls showed a small increase over time. Vaccinees had significantly higher levels than controls against class 1 and 3 porins and LPS in acute sera, against all antigens during early convalescence, and against class 1 and 3 porins in the later sera. Vaccinees who were infected with strains expressing subtype P1.7,16 proteins demonstrated a level of IgG binding to protein P1.7,16 with early-convalescent-phase sera that was fourfold higher than that of those infected with other strains. Bactericidal titers in serum against the vaccine strain were 192-fold higher for vaccinees than those for controls during early convalescence, but similarly low levels were found during late convalescence. A vaccine-induced anamnestic response of specific and functional antibody activities was thus shown, but the decrease in protection over time after vaccination indicated that two vaccine doses did not induce sufficient levels of long-term protective antibodies.Serogroup B meningococcal disease is a major health problem in many countries throughout the world. Serogroup B polysaccharide vaccine is poorly immunogenic in humans (66), probably because of its structural similarity to sialic acid residues on human cells (20). Therefore, vaccines based on noncapsular surface antigens have been developed and used in several trials (6, 7, 19, 21, 56).In Norway, the high incidence of meningococcal disease, which is caused mainly by B:15:P1.7,16 strains of the ET-5 complex (8, 12, 33), led to a placebo-controlled double-blind protection trial between 1988 and 1991. An outer membrane vesicle (OMV) vaccine from a representative epidemic strain (strain 44/76), which was adsorbed to aluminum hydroxide (24), was given in two doses to 88,800 secondary school students, while 83,000 received the placebo preparation of aluminum hydroxide. After 29 months of observation time, the point estimate for protection against group B meningococcal disease was 57.2% (P = 0.012) (6). From June 1991, the study continued as an open trial in which 49,000 of the previous placebo controls accepted vaccination (5). In this part of the trial, the 64,600 nonparticipants in the blinded part served as additional controls, since they were proven to have the same risk of contracting meningococcal disease as those given the placebo (5).In the blinded part of the trial, 12 vaccinees and 24 controls contracted systemic group B meningococcal disease. None of the survivors showed significant complement deficiencies (26, 30). During the first year of the open part (1991 to 1992), in which 137,800 vaccinees and 98,600 controls participated, the corresponding numbers with group B disease were 8 and 11, respectively (5). The latter vaccine failures had all been vaccinated in 1988 to 1989; a decrease in protection over time after vaccination was also observed in the blinded part of the trial (6, 50). From most of the patients, one or more serum samples were collected at different times after onset of disease. In the present study, digital image analysis was used to measure the immunoglobulin G (IgG) binding intensities of these sera to the major OMV vaccine antigens on immunoblots. The aim of our study was to compare the quantitated IgG responses of vaccine failures and unvaccinated controls and to analyze the possible associations between these antibody specificities and the bactericidal activities in serum. Our results demonstrated that the group B vaccine had induced immunological memory but that two doses were not sufficient to obtain long-term protective antibody levels. A preliminary immunoblot study of sera from nine of the patients has been published previously (59).     

Association of Cytokine Gene Polymorphisms in Patients with Tuberculosis and Their Household Contacts

Cytokine gene polymorphisms are known to be associated with functional differences in cytokine regulation and may affect host susceptibility to tuberculosis (TB). Contacts are important group in developing tuberculosis infection and are 10–60 times more likely to develop TB than general population. The present study was carried out in patients with TB (N = 176), their household contacts (HHC; N = 155) from Free Chest TB Clinic PPM DOTS (1TU) covering 500,000 population at Mahavir Hospital and Research Centre, Hyderabad, and healthy controls (HC; N = 170) also included. The association of single‐nucleotide polymorphisms (SNPs) in the promoter region of TNF‐α (?308G/A), IL‐2 (?330T/G), IL‐4 (?589C/T) and in exon region of TGF‐β1 (+869T/C) genes was assessed by ARMS & PCR‐RFLP using specific primers in the above‐mentioned subjects. The differences in allelic or genotypic frequencies of TNF‐α (?308G/A) between patients, their HHC and HC were not statistically significant (P > 0.05). IL‐2 (?330T/G) TG genotype was significantly different between patients, HHC compared to HC (P < 0.002, OR‐1.997, 95%CI‐1.260‐3.168, P < 0.03, OR‐1.602, 955CI‐1.003‐2.561).IL‐4 (?589C/T) CC genotype was significantly different between patients and HC (P < 0.03, OR‐1.791, 95%CI‐1.009‐3.189) as well as between HHC and HC at P < 0.0001, OR‐2.56, 95%CI‐1.448‐4.545. In addition, the TGF‐β 1 (+869T/C) TC genotype was significantly associated with susceptibility to tuberculosis in patients when compared against HC(P < 0.0001, OR‐3.416, 95%CI‐2.063‐5.670) and HHC (P < 0.0001, OR‐2.357, 95%CI‐1.439‐3.868), respectively.MDR analysis indicated that TT genotype of TGF‐β1 with TT and CT genotypes of IL‐4 showed high risk with GA, TT genotypes of TNF‐α, IL‐2, respectively. Our results suggest that IL‐2 (‐330T/G), IL‐4 (‐589 C/T) and TGF‐β1 (+869T/C) gene polymorphisms may be associated with TB susceptibility.