Characterization of invasive meningococcal disease case isolates in Atlantic Canada, 2014 to 2020: spatial-temporal variations of clones and predicted meningococcal B vaccine coverage.

Introduction. Invasive meningococcal disease (IMD) caused by Neisseria meningitidis may show temporal and geographical changes in both the epidemiology and the characteristics of the strains involved.Gap statement. A study that examined invasive N. meningitidis causing IMD in Atlantic Canada from 2009 to 2013 was published in 2014. Data from subsequent years have not been described.Aim. This study examined the molecular epidemiology of IMD in four Atlantic Provinces of Canada as well as potential serogroup B (MenB) vaccine coverage.Methods. Individual IMD case isolates recovered from 2014 to 2020 were analysed for serotype and serosubtype antigens as well as by whole-genome sequencing (WGS) for prediction of potential MenB vaccine coverage.Results. Of the 56 IMD isolates, 42, 8, 5 and 1 were MenB, serogroup Y, serogroup W (MenW) and serogroup C, respectively. Geographical differences in the distribution of MenB clones revealed concentration of sequence type (ST)-269 clonal complex (cc) and ST-60 cc in Newfoundland and Labrador, while ST-41/44 cc (particularly ST-154) was predominantly found in New Brunswick and Nova Scotia. Core genome multi-locus sequence typing (cgMLST) also separated the New Brunswick and Nova Scotia ST-154 isolates into two clusters, with differences in their nhba and penA alleles. Furthermore, cgMLST also separated the ST-269 cc isolates in Atlantic Canada into the ST-1611 and the ST-269/ST-8924 clusters, with the latter showing high similarity to the ST-269 that first emerged in the Province of Quebec. Genetic Meningococcal Antigen Typing System showed that 54.8 % of MenB were predicted to be covered by the MenB vaccine Bexsero, with a further 38.1 % potentially covered by virtue of the presence of genes that encoded factor H-binding protein variant 1 proteins. Meningococcal deduced vaccine antigen reactivity predicted from WGS data showed that 95.3 % of MenB were covered by Trumenba. Four cases of IMD due to MenW ST-11 cc were also identified, with the first case found in 2018.Conclusions. This study provided evidence concerning the dynamics of N. meningitidis strains causing IMD in Atlantic Canada, with both geographical and temporal differences found. MenB vaccine appeared to provide good coverage of MenB IMD, especially towards the predominant strain of ST-154.

Culture-Confirmed Invasive Meningococcal Disease in Canada, 2010 to 2014: Characterization of Serogroup B Neisseria meningitidis Strains and Their Predicted Coverage by the 4CMenB Vaccine.

The molecular epidemiology of culture-confirmed invasive meningococcal disease (IMD) in Canada from 2010 to 2014 was studied with an emphasis on serogroup B Neisseria meningitidis (MenB) isolates, including their predicted coverage by the 4CMenB vaccine. The mean annual incidence rates of culture confirmed IMD varied from 0.19/100,000 in Ontario to 0.50/100,000 in New Brunswick and 0.59/100,000 in Quebec. In both Quebec and Atlantic region, MenB was significantly more common than other serogroups, while in other provinces, both MenB and serogroup Y (MenY) were almost equally common. The majority of MenB cases (67.0%) were in those aged ≤24 years, while most MenC (75.0%) and MenY (69.6%) cases were in adults more than 24 years old. The 349 MenB isolates were grouped into 103 sequence types (STs), 90 of which belonged to 13 clonal complexes (CCs). A large number of 4CMenB antigen genes were found among the Canadian MenB, which is predicted to encode 50 factor H binding protein (fHbp) types, 40 NHBA types, and 55 PorA genotypes. Provinces and regions were found to have their own unique MenB STs. A meningococcal antigen typing system assay predicted an overall MenB coverage by 4CMenB to be 73.6%, with higher coverage predicted for the two most common STs: 100% for ST154 and 95.9% for ST269, leading to higher coverage in both the Atlantic region and Quebec. Higher coverage (81.4%) was also found for MenB recovered from persons aged 15 to 24 years, followed by strains from infants and children ≤4 years old (75.2%) and those aged 5 to 14 years (75.0%).IMPORTANCE Laboratory surveillance of invasive meningococcal disease (IMD) is important to our understanding of the evolving nature of the Neisseria meningitidis strain types causing the disease and the potential coverage of disease strains by the newly developed vaccines. This study examined the molecular epidemiology of culture-confirmed IMD cases in Canada by examining the strain types and the potential coverage of a newly licensed 4CMenB vaccine on Canadian serogroup B N. meningitidis strains. The strain types identified in different parts of Canada appeared to be unique as well as their predicted coverage by the 4CMenB vaccine. These data were compared to data obtained from previous studies done in Canada and elsewhere globally. For effective control of IMD, laboratory surveillance of this type was found to be essential and useful to understand the dynamic nature of this disease.

Identification and Characterization of "Haemophilus quentini" Strains Causing Invasive Disease in Ontario, Canada (2016 to 2018).

Haemophilus influenzae is a well-established human pathogen capable of causing a range of respiratory and invasive diseases. Since the 1970s, it has been observed that a nontypeable cryptic genospecies of H. influenzae, most often biotype IV, has been associated with the genitourinary tracts of females and with invasive neonatal infections. This distinct genospecies has been provisionally named "Haemophilus quentini" Here, we report seven cases of invasive H. quentini disease in patients from Ontario, Canada, over a 2-year period. Significantly, while most reports of invasive disease with H. quentini to date have been in neonates, we observed five cases in adults (three in women of childbearing age and two in seniors) as well as two in neonates. Identification of H. quentini is challenging and was not possible for frontline laboratories, requiring work at the reference laboratory level. We describe in detail the biochemical results, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-Tof MS) results, and PCR results with several targets, including the 16S rRNA gene and multilocus sequence typing (MLST) genes, for the seven Ontario H. quentini isolates and several controls. Our data, combined with those of other publications, support the fact that H. quentini is distinct from H. influenzae and Haemophilus haemolyticus This organism is recognized as a pathogen of neonates, but we hypothesize that it may be underrecognized as an important pathogen in adults as well, particularly pregnant women. By sharing the detailed descriptions of these isolates, we hope to enable other laboratories to better identify H. quentini so that the true prevalence of this organism and disease can be explored.

Ciprofloxacin-resistant Neisseria meningitidis in Canada: likely imported strains.

The prevalence of ciprofloxacin-resistant Neisseria meningitidis in Canada was studied by testing 346 isolates received at the National Microbiology Laboratory during the calendar years 2013 to 2015. Of the 277 individual invasive and 69 noninvasive isolates tested, only 2 serogroup C (MenC) isolates were found to be resistant to ciprofloxacin. Both MenC were typed as sequence type (ST)-4821, a unique clone found mainly in China, thus suggesting both isolates might be from travel-related or imported cases. This prompted us to also examine 6 serogroup A (MenA) isolates in our collection, since MenA is not currently endemic in Canada. Three MenA from 2006 were resistant to ciprofloxacin and they were typed as ST-4789. A ciprofloxacin-resistant MenA strain of ST-4789 was responsible for a meningococcal disease outbreak in Delhi, India, in 2005 to 2006. The 2 MenC and 3 MenA ciprofloxacin-resistant N. meningitidis were from patients residing in British Columbia.

Impact of an Immunization Campaign to Control an Increased Incidence of Serogroup B Meningococcal Disease in One Region of Quebec, Canada.

Background: Invasive meningococcal disease (IMD) incidence increased in Quebec, starting in 2003, and was caused by a serogroup B sequence type 269 clone. The Saguenay-Lac-Saint-Jean (SLSJ) region was particularly affected with a rate of 3.4 per 100000 person-years in 2006-2013. In May 2014, an immunization campaign was launched in SLSJ, using the 4-component protein-based meningococcal vaccine (MenB-4C). We aimed to evaluate the impact of the campaign 2 years after its initiation. Methods: Immunization registry data and serogroup B invasive meningococcal disease (B-IMD) cases notified to public health authorities and confirmed by culture or polymerase chain reaction from July 1996 to December 2016 were analyzed, including a multivariate Poisson regression model of incidence rates. Results: By the end of the campaign, 82% of the 59000 targeted SLSJ residents between 2 months and 20 years of age had been immunized. Following the initiation of the campaign, no B-IMD case occurred among vaccinees, whereas 2 cases were reported among unvaccinated adult SLSJ residents, and a third case in an unvaccinated child who had stayed in the region during the week prior to disease onset, in 2015. B-IMD incidence decreased in all other regions in the years 2015-2016 but sporadic cases continued to occur. A multivariate analysis showed a significant effect of the campaign in the SLSJ region (relative B-IMD risk: 0.22; P = .04). Conclusions: Results suggest a high level of protection provided by MenB-4C following mass vaccination at regional level. This, along with reassuring safety data, supports the current recommendations for MenB-4C use for controlling outbreaks caused by clones covered by the vaccine.

Characterization of invasive Neisseria meningitidis strains from Québec, Canada, during a period of increased serogroup B disease, 2009-2013: phenotyping and genotyping with special emphasis on the non-carbohydrate protein vaccine targets.

BACKGROUND: The epidemiology of invasive meningococcal disease (IMD) in Québec, Canada, has been dominated in the past decade by a clone of serogroup B (MenB) Neisseria meningitidis defined by multi-locus sequence typing (MLST) as sequence type (ST)-269. With the licensure of a new MenB vaccine Bexsero (4CMenB) in Canada, this study characterized invasive N. meningitidis recovered in Québec from 2009 to 2013, with an objective to examine the diversity of the 4CMenB vaccine antigens. Isolates were serogrouped by antisera and genogrouped by PCR, and further typed by whole cell ELISA for serotype and serosubtype antigens. Clonal analysis was done by MLST. Isolates were genotyped by analysis of their 4CMenB vaccine antigen genes of PorA, factor H binding protein (fHbp), Neisserial Heparin Binding Antigen (NHBA), and Neisseria Adhesin A (NadA). RESULTS: Of the 263 IMD isolates analysed, 229, 16, 10, 7, and 1 belonged to MenB, MenY, MenW, MenC, and MenX, respectively. Of the 229 MenB, 159 (69.4 %) were typed as ST-269 clonal complex (CC); and they possessed a restricted number of three fHbp and five nhba gene alleles. Nine N. meningitidis isolates (eight MenB and one MenY) were found to possess at least one gene that encoded for an antigen that matched exactly with protein variants in the 4CMenB vaccine. Two MenB expressed PorA antigen P1.4 and possessed the nhba gene for peptide 2; four other MenB were predicted to have NHBA peptide 2; another two MenB were predicted to encode fHbp peptide 1.1; and a single MenY was found to have nadA gene for NadA peptide 8. In addition, another 172 isolates were found to possess genes for variant 1 fHbp peptides other than peptide 1.1 or NadA variant 1-2/3 peptides other than peptide 8; and therefore, may potentially be covered by 4CMenB. CONCLUSION: The most prevalent clone of N. meningitidis in Quebec was ST-269 CC; and 96 % of the isolates in this CC were predicted to be covered by 4CMenB vaccine. Extensive genetic diversity was found in the other IMD isolates in Québec which might suggest a lower coverage by the vaccine when compared to the ST-269 MenB.

Characterization of invasive Neisseria meningitidis from Atlantic Canada, 2009 to 2013: With special reference to the nonpolysaccharide vaccine targets (PorA, factor H binding protein, Neisseria heparin-binding antigen and Neisseria adhesin A).

BACKGROUND: Serogroup B Neisseria meningitidis (MenB) has always been a major cause of invasive meningococcal disease (IMD) in Canada. With the successful implementation of a meningitis C conjugate vaccine, the majority of IMD in Canada is now caused by MenB. OBJECTIVE: To investigate IMD case isolates in Atlantic Canada from 2009 to 2013. Data were analyzed to determine the potential coverage of the newly licensed MenB vaccine. METHODS: Serogroup, serotype and serosubtype antigens were determined from IMD case isolates. Clonal analysis was performed using multilocus sequence typing. The protein-based vaccine antigen genes were sequenced and the predicted peptides were investigated. RESULTS: The majority of the IMD isolates were MenB (82.5%, 33 of 40) and, in particular, sequence type (ST)-154 B:4:P1.4 was responsible for 47.5% (19 of 40) of all IMD case isolates in Atlantic Canada. Isolates of this clone expressed the PorA antigen P1.4 and possessed the nhba genes encoding for Neisseria heparin-binding antigen peptide 2, which together matched exactly with two of the four components of the new four-component meningococcal B vaccine. Nineteen MenB isolates had two antigenic matches, another five MenB and one meningitis Y isolate had one antigenic match. This provided 75.8% (25 of 33) potential coverage for MenB, or a 62.5% (25 of 40) overall potential coverage for IMD. CONCLUSION: From 2009 to 2013, IMD in Atlantic Canada was mainly caused by MenB and, in particular, the B:4:P1.4 ST-154 clone, which accounted for 47.5% of all IMD case isolates. The new four-component meningococcal B vaccine appeared to offer adequate coverage against MenB in Atlantic Canada.

HISTORIQUE: Le Neisseria meningitidis du sérogroupe B (MenB) a toujours été une cause importante de méningococcie invasive (MI) au Canada. Depuis l'adoption d'un vaccin conjugué contre le méningocoque du groupe C, la majorité des MI au Canada sont désormais attribuables au MenB. OBJECTIF: Examiner les isolats de cas de MI dans les Maritimes entre 2009 et 2013. Analyser les données pour déterminer la couverture potentielle du vaccin nouvellement homologué contre le MenB. MÉTHODOLOGIE: Les chercheurs ont déterminé le sérogroupe, le sérotype et les antigènes des sous-types sérologiques des isolats de cas de MI. Ils ont effectué l'analyse clonale au moyen du typage génomique multilocus. Ils ont séquencé les gènes des antigènes du vaccin à base de protéines et examiné les peptides prédits. RÉSULTATS: La majorité des isolats de MI étaient des MenB (82,5 %, 33 sur 40). Notamment, le type séquentiel (TS)-154 B:4:P1,4 était responsable de 47,5 % (19 sur 40) de tous les isolats de cas de MI dans les Maritimes. Les isolats de ce clone ont exprimé l'antigène porA P1.4 et étaient dotés des gènes nhba codant pour le peptide 2 de l'antigène de liaison à l'héparine de Neisseria. Ensemble, ces antigènes correspondaient exactement à deux des quatre composants du nouveau vaccin contre le méningocoque du groupe B à quatre composants. Dix-neuf isolats du MenB étaient dotés de deux correspondances antigéniques, tandis que cinq autres MenB et un isolat de la méningite Y étaient dotés d'une correspondance antigénique. Ces résultats assuraient une couverture potentielle du MenB de 72,7 % (24 sur 33) ou une couverture potentielle globale de la MI de 62,5 % (25 sur 40). CONCLUSION: De 2009 à 2013, dans les Maritimes, la MI était surtout causée par le MenB, en particulier le clone B:4:P1.4 ST-154, responsable de 47,5 % de tous les isolats de cas de MI. Le nouveau vaccin contre le méningocoque du groupe B à quatre composants semble offrir une couverture pertinente contre le MenB dans cette région.

Determination of serotyping antigens, clonal analysis and genetic characterization of the 4CMenB vaccine antigen genes in invasive Neisseria meningitidis from Western Canada, 2009 to 2013.

This study examined invasive Neisseria meningitidis recovered from invasive meningococcal disease (IMD) cases in Western Canada between 2009 and 2013. A total of 161 isolates from individual IMD cases were analysed for serogroup, serotype, serosubtype, PorA genotype, multi-locus sequence type and nucleotide sequence of their 4CMenB vaccine antigen genes. Sixty-nine isolates were serogroup B (MenB), 47 were serogroup Y (MenY), 22 were serogroup C (MenC), 19 were serogroup W (MenW), three were serogroup E and one was non-encapsulated. MenC, MenY and MenW were mainly clonal, represented primarily by clonal complex (cc) 11, cc23 or cc167, and cc22, respectively. In contrast, MenB were composed of eight different ccs together with 11 isolates not assigned to any known cc. Antigenic analysis and PorA genotyping confirmed the heterogeneity of MenB isolates, while such results supported the clonal nature of most MenC, MenY and MenW isolates. Thirty-four (21.1%) isolates had at least one gene that encoded one matching vaccine protein component of the 4CMenB vaccine (i.e. PorA P1.4; fHbp variant 1.1; NHBA peptide 2; and NadA-1, -2, or -3). An additional 18 isolates had genes that encoded variant 1 or subfamily B factor H binding proteins of this same vaccine.

Population genetics of Haemophilus influenzae serotype a in three Canadian provinces.

Haemophilus influenzae serotype a (Hia) is an important pathogen since the introduction of vaccines for control of disease due to serotype b strains. Using a sodC-based polymerase chain reaction, Hia can be divided into 2 phylogenetic divisions, each with their own unique multilocus sequence types. Most Canadian Hia belongs to clonal division I and the ST-23 clonal complex. The recently described hypervirulent clone of ST-4 was found in a single Canadian isolate. Therefore, surveillance of invasive H. influenzae disease should include serotyping to detect Hia and multilocus sequence typing to detect hypervirulent clones.

Real-time polymerase chain reaction for detection of encapsulated Haemophilus influenzae using degenerate primers to target the capsule transport gene bexA.

A real-time polymerase chain reaction assay that uses degenerate primers and a dual-labelled probe was developed to detect the bexA gene of Haemophilus influenzae, including those belonging to non-b serotypes as well as clonal division II strains. This assay is sensitive and specific, detecting 20 copies of the gene, but negative with a variety of bacteria associated with meningitis and bacteremia or septicemia.

Genetic and antigenic characterization of invasive endemic serogroup B Neisseria meningitidis from Ontario, Canada, in 2001-2010.

This study examined the antigenic and genetic diversity of serogroup B Neisseria meningitidis (MenB) recovered from invasive meningococcal disease (IMD) cases in Ontario, Canada, over the period 2001-2010 during which no MenB outbreaks had occurred. MenB was found to be responsible for 39 % of all IMD cases, with the remaining cases caused mainly by serogroups Y (28 %), C (23.5 %) and W135 (8 %). One hundred and ninety-three individual MenB case isolates were collected and characterized. Of the 88 sequence types (STs) identified, 75 were grouped into 14 known clonal complexes (CCs), whilst 13 STs were not assigned to any known CC. Fifty-seven different PorA genotypes and 88 STs defined the diversity of invasive MenB in Ontario, which supported the endemic nature of MenB disease in Ontario. Despite the presence of the hypervirulent ST-41/44 and ST-32 CCs, no single ST was predominant and responsible for a large number of IMD cases. Although the Québec outbreak clone of ST-269 was also found in Ontario, the 20 case isolates were genetically diverse: they grouped into seven STs and did not have a predominant PorA genotype. eburst analysis identified a new CC responsible for 14.5 % of the MenB case isolates. The six most common PorA variable region 2 (VR2) genotypes (VR2-9, -4, -14, -16, -13-1 and -16-3) were found in 67 % of invasive MenB isolates.

Invasive potential of nonencapsulated disease isolates of Neisseria meningitidis.

The capsule of Neisseria meningitidis is the major virulence factor that enables this bacterium to overcome host immunity elicited by complement and phagocytes, rendering it capable of surviving in blood. As such, nonencapsulated N. meningitidis isolates are generally considered nonpathogenic. Here, we consider the inherent virulence of two nonencapsulated N. meningitidis isolates obtained from our national surveillance of infected blood cultures in Canada. Capsule deficiency of both strains was confirmed by serology and PCR for the ctrA to ctrD genes and siaA to siaC genes, as well as siaD genes specific to serogroups B, C, Y, and W135. In both strains, the capsule synthesis genes were replaced by the capsule null locus, cnl-2. In accordance with a lack of capsule, both strains were fully susceptible to killing by both human and baby rabbit complement. However, in the presence of cytidine-5' monophospho-N-acetylneuraminic acid (CMP-NANA), allowing for lipooligosaccharide (LOS) sialylation, a significant increase of resistance to complement killing was observed. Mass spectrometry of purified LOS did not reveal any uncommon modifications that would explain their invasive phenotype. Finally, in a mouse intraperitoneal challenge model, these nonencapsulated isolates displayed enhanced virulence relative to an isogenic mutant of serogroup B strain MC58 lacking capsule (MC58ΔsiaD). Virulence of all nonencapsulated isolates tested was below that of encapsulated serogroup B strains MC58 and B16B6. However, whereas no mortality was observed with MC58ΔsiaD, 5/10 mice succumbed to infection with strain 2275 and 2/11 mice succumbed to strain 2274. Our results suggest the acquisition of a new virulence phenotype by these nonencapsulated strains.

Invasive serogroup B Neisseria meningitidis in Quebec, Canada, 2003 to 2010: persistence of the ST-269 clone since it first emerged in 2003.

In the era after the introduction of the meningococcal serogroup C conjugate vaccine, from 1 January 2003 to 31 December 2010, serogroup B meningococci were the major cause of invasive meningococcal disease in the province of Québec, Canada, being responsible for 72% of all meningococcal disease cases. Of the 334 invasive serogroup B Neisseria meningitidis strains analyzed, 53.9% belonged to the ST-269 clonal complex (CC). Since it first emerged in 2003, the percentage of invasive serogroup B isolates that belonged to the ST-269 CC had increased from 35% in 2003 to 76% in 2010. Among the 180 meningococci in the ST-269 CC, 91.7% belonged to a single ST (ST-269). The most common PorA genotypes identified in the ST-269 CC were (i) VR1 19-1, VR2 15-11, VR3 36 (84%) and (ii) VR1 18-7, VR2 9, VR3 35-1 (9%). Cases of invasive disease due to the ST-269 CC were commonly found in those aged 11 to 19 years (30.5%) and 20 to 40 years (25.5%). Meningococci of the ST-269 CC were uncommon in other Canadian provinces. In contrast to the ST-269 CC, invasive serogroup B meningococci that belonged to the ST-41/44 CC were much more diverse genetically. However, one ST (ST-571), which is uncommon in the United States, accounted for 35% of all cases due to this CC. The current finding suggests that the ST-269 clone may indeed represent an emerging hypervirulent clone of meningococci.

Nonencapsulated or nontypeable Haemophilus influenzae are more likely than their encapsulated or serotypeable counterparts to have mutations in their fucose operon.

Population biology of Haemophilus influenzae can be studied by multilocus sequence typing (MLST), and isolates are assigned sequence types (STs) based on nucleotide sequence variations in seven housekeeping genes, including fucK. However, the ST cannot be assigned if one of the housekeeping genes is absent or cannot be detected by the current protocol. Occasionally, strains of H. influenzae have been reported to lack the fucK gene. In this study, we examined the prevalence of this mutation among our collection of H. influenzae isolates. Of the 704 isolates studied, including 282 encapsulated and 422 nonencapsulated isolates, nine were not typeable by MLST owing to failure to detect the fucK gene. All nine fucK-negative isolates were nonencapsulated and belonged to various biotypes. DNA sequencing of the fucose operon region confirmed complete deletion of genes in the operon in seven of the nine isolates, while in the remaining two isolates, some of the genes were found intact or in parts. The significance of these findings is discussed.

Reducing lambda repressor to the core.

Lambda repressor fragment λ(*)(6-85) is one of the fastest folding small protein fragments known to date. We hypothesized that removal of three out of five helices of λ(*)(6-85) would further reduce this protein to its smallest folding core. Molecular dynamics simulations singled out two energetically stable reduced structures consisting of only helices 1 and 4 connected by a short glycine/serine linker, as well as a less stable control. We investigated these three polypeptides and their fragments experimentally by using circular dichroism, fluorescence spectroscopy, and temperature jump relaxation spectroscopy to gain insight into their thermodynamic and kinetic properties. Based on the thermal melts, the order of peptide stability was in correspondence with theoretical predictions. The most stable two-helix bundle, λ(blue1), is a cooperatively folding miniprotein with the same melting temperature and folding rate as the full-length λ(*)(6-85) pseudo wild type and a well-defined computed structure.

Invasive Haemophilus influenzae in British Columbia: non-Hib and non-typeable strains causing disease in children and adults.

OBJECTIVES: To characterize invasive Haemophilus influenzae and to examine the population at risk for invasive H. influenzae disease in British Columbia, Canada, 2008-2009. METHODS: H. influenzae recovered from individual patients were characterized by serotyping, biotyping, multilocus sequence typing (MLST), and antibiotic susceptibility testing. Age information was recorded from specimen requisition forms. RESULTS: Of the 98 cases, 66% were caused by non-typeable strains, followed by serotypes b (12%), a (10%), f (10%), and e (1%). Cases caused by serotypes b and f and non-typeable strains were mainly in adults over 18 years of age, while cases due to serotype a were mainly in children under the age of 2 years. Different sequence types were found in encapsulated strains according to their serotypes, and non-typeable strains had their own unique sequence types. No capsule switching was documented. Antibiotic resistance was common among non-typeable strains, with 31% identified as genotypic ß-lactamase-negative ampicillin-resistant (BLNAR) strains. CONCLUSION: Invasive H. influenzae disease in a population vaccinated against Hib was age-dependent and involved both non-typeable and encapsulated strains. Adults were susceptible to invasive diseases due to non-typeable and serotype b and f strains, while in children, most diseases were due to serotype a bacteria.

Characterization of nontypeable Haemophilus influenzae collected from respiratory infections and invasive disease cases in Manitoba, Canada.

With the introduction of the Haemophilus influenzae serotype b (Hib) vaccine, invasive Hib disease has decreased substantially, but nontypeable H. influenzae (NT Hi) disease appears to be increasing. In order to understand the origin of NT Hi strains and their relationship with serotypeable strains, we analysed 125 NT Hi isolates collected from individual patients with either invasive disease (70 isolates) or respiratory tract infections (55 isolates). Serotype-specific and capsular transport genes were absent by PCR analysis, confirming their nonencapsulated status, which also suggested the NT Hi isolates were not encapsulated strains that shed their capsules. Multilocus sequence typing confirmed the NT Hi isolates did not have the same genetic background as serotypeable strains, including Hib. Despite the genetic heterogeneity found, two major genetic clusters were identified, both containing invasive and respiratory isolates. Fourteen invasive isolates and nine respiratory isolates produced beta-lactamase and were ampicillin resistant. More invasive (26.8%) than respiratory isolates (10.9%) showed decreased susceptibility towards ampicillin by a mechanism unrelated to beta-lactamase production. Besides a change in the capsule status of invasive Hi strains, the burden of invasive Hi disease, which used to be mainly a childhood disease, has now shifted to involve both adults and infants.

Genetic detection of quinolone resistance in Haemophilus parainfluenzae: Mutations in the quinolone resistance-determining regions of gyrA and parC.

The quinolone resistance-determining regions of gyrA and parC of both quinolone-sensitive and quinolone-resistant Haemophilus parainfluenzae strains were amplified and sequenced. Similar to Haemophilus influenzae, resistance to quinolones in H parainfluenzae is associated with mutations in the quinolone resistance-determining regions of both gyrA and parC. The present study discusses the importance of this finding.

Immunochemical studies and genetic background of two Neisseria meningitidis isolates expressing unusual capsule polysaccharide antigens with specificities of both serogroup Y and W135.

We described 2 unusual Neisseria meningitidis strains isolated from epidemiologically unrelated invasive meningococcal disease cases in Ontario, Canada. Both isolates have features typical of serogroup Y N. meningitidis: are of serotype 2c, are of the multi-locus sequence types typical of the serogroup Y strains in Canada, and are genotyped as serogroup Y based on a previously described PCR-ELISA method that detects the serogroup-Y-specific siaD gene. However, both strains were poly-agglutinable in both anti-Y and anti-W135 antisera. Further studies on 1 of these 2 isolates showed the presence of glucose and galactose as well as sialic acids in its purified capsular polysaccharide, suggesting the presence of both serogroup Y and serogroup W135 polysaccharides. Rabbit antisera produced to this strain contained antibodies to both purified serogroup Y and serogroup W135 capsular polysaccharides. Absorption experiments with either serogroup Y or serogroup W135 bacteria confirmed the presence of antibodies to these 2 different polysaccharides. DNA sequencing of the cps operon from both isolates revealed a siaD gene with 99.7% homology to the published siaD sequence from a serogroup Y strain but with 3 point mutations that all resulted in amino acid changes. How these strains may affect results of routine surveillance, PCR diagnosis, and immuno-protection by vaccination are discussed.