Evaluation of the World Health Organization Global Invasive Bacterial Vaccine-Preventable Disease (IB-VPD) Surveillance Network's Laboratory External Quality Assessment Programme, 2014-2019.

Introduction. In 2009, the World Health Organization (WHO) established the Global Invasive Bacterial Vaccine Preventable Disease (IB-VPD) Surveillance Network (GISN) to monitor the global burden and aetiology of bacterial meningitis, pneumonia and sepsis caused by Haemophilus influenzae (Hi), Neisseria meningitidis (Nm) and Streptococcus pneumoniae (Sp).Hypothesis/Gap Statement. The GISN established an external quality assessment (EQA) programme for the characterization of Hi, Nm and Sp by culture and diagnostic PCR.Aim. To assess the performance of sentinel site laboratories (SSLs), national laboratories (NLs) and regional reference laboratories (RRLs) between 2014 and 2019 in the EQA programme.Methodology. Test samples consisted of bacterial smears for Gram-staining, viable isolates for identification and serotyping or serogrouping (ST/SG), plus simulated cerebrospinal fluid (CSF) samples for species detection and ST/SG by PCR. SSLs and NLs were only required to analyse the slides for Gram staining and identify the species of the live isolates. RRLs, and any SLs and NLs that had the additional laboratory capacity, were also required to ST/SG the viable isolates and analyse the simulated CSF samples.Results. Across the period, 69-112 SS/NL labs and eight or nine RRLs participated in the EQA exercise. Most participants correctly identified Nm and Sp in Gram-stained smears but were less successful with Hi and other species. SSLs/NLs identified the Hi, Nm and Sp cultures well and also submitted up to 56 % of Hi, 62 % of Nm and 33 % of Sp optional ST/SG results each year. There was an increasing trend in the proportion of correct results submitted over the 6 years for Nm and Sp. Some SSLs/NLs also performed the optional detection and ST/SG of the three organisms by PCR in simulated CSF from 2015 onwards; 89-100 % of the CSF samples were correctly identified and 76-93 % of Hi-, 90-100 % of Nm- and 75-100 % of Sp-positive samples were also correctly ST/SG across the distributions. The RRLs performed all parts of the EQA to a very high standard, with very few errors across all aspects of the EQA.Conclusion. The EQA has been an important tool in maintaining high standards of laboratory testing and building of laboratory capacity in the GISN.

Assessment of trimethoprim-sulfamethoxazole susceptibility testing methods for fastidious Haemophilus spp.

OBJECTIVES: To compare the determinants of trimethoprim-sulfamethoxazole resistance with established susceptibility values for fastidious Haemophilus spp., to provide recommendations for optimal trimethoprim-sulfamethoxazole measurement. METHODS: We collected 50 strains each of Haemophilus influenzae and Haemophilus parainfluenzae at Bellvitge University Hospital. Trimethoprim-sulfamethoxazole susceptibility was tested by microdilution, E-test and disc diffusion using both Mueller-Hinton fastidious (MH-F) medium and Haemophilus test medium (HTM) following EUCAST and CLSI criteria, respectively. Mutations in folA, folP and additional determinants of resistance were identified in whole-genome-sequenced isolates. RESULTS: Strains presented generally higher rates of trimethoprim-sulfamethoxazole resistance when grown on HTM than on MH-F, independent of the methodology used (average MIC 2.6-fold higher in H. influenzae and 1.2-fold higher in H. parainfluenzae). The main resistance-related determinants were as follows: I95L and F154S/V in folA; 3- and 15-bp insertions and substitutions in folP; acquisition of sul genes; and FolA overproduction potentially linked to mutations in -35 and -10 promoter motifs. Of note, 2 of 19 H. influenzae strains (10.5%) and 9 of 33 H. parainfluenzae strains (27.3%) with mutations and assigned as resistant by microdilution were inaccurately considered susceptible by disc diffusion. This misinterpretation was resolved by raising the clinical resistance breakpoint of the EUCAST guidelines to ≤30 mm. CONCLUSIONS: Given the routine use of disc diffusion, a significant number of strains could potentially be miscategorized as susceptible to trimethoprim-sulfamethoxazole despite having resistance-related mutations. A simple modification to the current clinical resistance breakpoint given by the EUCAST guideline for MH-F ensures correct interpretation and correlation with the reference standard method of microdilution.

Intranasal coinfection model allows for assessment of protein vaccines against nontypeable Haemophilus influenzae in mice.

PURPOSE: Nontypeable Haemophilus influenzae (NTHi) is a commensal in the human nasopharynx and the cause of pneumonia, meningitis, sinusitis, acute exacerbations of chronic obstructive pulmonary disease and acute otitis media (AOM). AOM is the most common ailment for which antibiotics are prescribed in the United States. With the emergence of new strains of antibiotic-resistant bacteria, finding an effective and broad coverage vaccine to protect against AOM-causing pathogens has become a priority. Mouse models are a cost-effective and efficient way to help determine vaccine efficacy. Here, we describe an NTHi AOM model in C57BL/6J mice, which also utilizes a mouse-adapted H1N1 influenza virus to mimic human coinfection. METHODOLOGY: We tested our coinfection model using a protein vaccine formulation containing protein D, a well-studied NTHi vaccine candidate that can be found in the 10-valent Streptococcus pneumoniae conjugate vaccine. We verified the usefulness of our mouse model by comparing bacterial loads in the nose and ear between protein D-vaccinated and control mice. RESULTS: While there was no measurable difference in nasal bacterial loads, we did detect significant differences in the bacterial loads of ear washes and ear bullae between vaccinated and control mice. CONCLUSION: The results from this study suggest that our NTHi AOM coinfection model is useful for assessing protein vaccines.

Molecular characterization of fluoroquinolones, macrolides, and imipenem resistance in Haemophilus influenzae: analysis of the mutations in QRDRs and assessment of the extent of the AcrAB-TolC-mediated resistance.

The aims of the present study were to characterize the mechanisms of resistance to fluoroquinolones, macrolides, and imipenem in Haemophilus influenzae, to assess the extent of the AcrAB-TolC-mediated resistance, and to define a core genome multilocus sequence typing (cgMLST) scheme for H. influenzae by using whole-genome sequencing. Four amino acid substitutions in GyrA (at Ser84 and Asp88), ParC (at Ser84), and ParE (at Asp420) were found to be closely associated to the MICs. We did not find any amino acid substitution surrounding the three highly conserved amino acid motifs in PBP3 related to imipenem resistance. All the isolates possessed the ermB gene. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) decreased the MIC of imipenem by twofold for FQR-6 and fourfold for GE47 and GE88 strains. For erythromycin, the MICs were decreased by twofold. We found that the six FQR isolates were clustered in two groups. The number of different loci within FQR-1_FQR-3_FQR-5 cluster was 6, while FQR-2 and FQR-4 differed for 21 loci. FQR-1_FQR-3_FQR-5 and FQR-2_FQR-4 clusters were distant among each other and compared to 19 genomes downloaded from NCBI, to 8 strains heteroresistant to imipenem, and to 4 strains monoresistant to ciprofloxacin isolated in Denmark. We confirmed that specific amino acid substitutions in GyrA, ParC, and ParE are implicated in quinolone resistance. Additionally, the degree of resistance is related to the number of these amino acid substitutions. We provide robust evidence that drug efflux is one of the substantial mechanisms of imipenem and erythromycin resistance in H. influenzae.

TEM-1-encoding small plasmids impose dissimilar fitness costs on Haemophilus influenzae and Haemophilus parainfluenzae.

Only two beta-lactamases, TEM-1 and ROB-1, have been observed in Haemophilus influenzae, while four different TEM but no ROB enzymes have been found in Haemophilus parainfluenzae. In order to investigate the mechanisms behind the dissemination of small beta-lactamase-encoding plasmids in H. influenzae and H. parainfluenzae, we assessed the fitness cost of three TEM-1- (pPN223, pA1209, pA1606), one TEM-15- (pSF3) and one ROB-1-bearing (pB1000) plasmid when expressed in either bacterial species. All plasmids were stable in H. influenzae and H. parainfluenzae except pB1000, which showed on average (sample mean) 76% curing in H. parainfluenzae after 5  days of subculture. Competition assays between isogenic strains with and without plasmid showed no competitive disadvantage of pPN223 and pA1606 in H. influenzae, or of pA1209 in H. parainfluenzae. In contrast, pSF3 and pB1000 were associated with significant competitive disadvantages in both species. Some of the competitive disadvantages may be related to differences in plasmid copy number and mRNA expression of the beta-lactamase genes, as revealed by quantitative PCR analysis. In conclusion, plasmids encoding TEM beta-lactamases isolated from H. influenzae and H. parainfluenzae can be stably transferred between species. The fast curing of pB1000 in H. parainfluenzae observed in this study correlates to the fact that ROB-1 has never been reported for this species. TEM-1-encoding plasmids are associated with the lowest level of fitness cost, but different TEM-1 plasmids confer different levels of fitness cost on the two hosts.

Investigating long range correlation in DNA sequences using significance tests of conditional mutual information.

This study exploits the use of Markov chain order estimation from symbol sequences of systems exhibiting long memory or long range correlations (LRC), such as DNA sequences. In the presence of limited sequence length, LRC chain can be approximated by a high order Markov chain. For the order estimation, the parametric significance test of conditional mutual information IC(m) is applied, found in an earlier work to be suitable for high order estimation. Here, it is computationally optimized applying an iterative algorithm for calculating IC(m) at increasing order m, enabling the analysis of long symbol sequences of high Markov chain order or LRC. The simulation study shows that when the true order is reasonably small, the estimated order saturates at the true order with the increase of the symbol sequence length, while when the true order is very large or the chain has LRC, the estimated order increases logarithmically with the symbol sequence length. The order estimation shows a different dependence on the DNA sequence length for bacteria, the plant Arabidopsis thaliana and the human chromosome, indicating a different long memory structure in their DNA.

Haemophilus influenzae clinical isolates with plasmid pB1000 bearing blaROB-1: fitness cost and interspecies dissemination.

Plasmid pB1000 is a mobilizable replicon bearing the bla(ROB-1) beta-lactamase gene that we have recently described in Haemophilus parasuis and Pasteurella multocida animal isolates. Here we report the presence of pB1000 and a derivative plasmid, pB1000', in four Haemophilus influenzae clinical isolates of human origin. Pulsed-field gel electrophoresis showed unrelated patterns in all strains, indicating that the existence of pB1000 in H. influenzae isolates is not the consequence of clonal dissemination. The replicon can be transferred both by transformation and by conjugation into H. influenzae, giving rise to recipients resistant to ampicillin and cefaclor (MICs, > or =64 microg/ml). Stability experiments showed that pB1000 is stable in H. influenzae without antimicrobial pressure for at least 60 generations. Competition experiments between isogenic H. influenzae strains with and without pB1000 revealed a competitive disadvantage of 9% per 10 generations for the transformant versus the recipient. The complete nucleotide sequences of nine pB1000 plasmids from human and animal isolates, as well as the epidemiological data, suggest that animal isolates belonging to the Pasteurellaceae act as an antimicrobial resistance reservoir for H. influenzae. Further, since P. multocida is the only member of this family that can colonize both humans and animals, we propose that P. multocida is the vehicle for the transport of pB1000 between animal- and human-adapted members of the Pasteurellaceae.

Long simple sequence repeats in host-adapted pathogens localize near genes encoding antigens, housekeeping genes, and pseudogenes.

Simple sequence repeats (SSRs) in DNA sequences are tandem iterations of a single nucleotide or a short oligonucleotide. SSRs are subject to slipped-strand mutations and a common source of phase variation in bacteria and antigenic variation in pathogens. Significantly long SSRs are generally rare in prokaryotic genomes, and long SSRs composed of iterations of mono-, di-, tri-, and tetranucleotides are mostly restricted to host-adapted pathogens. We present new results concerning associations between long SSRs and genes related to different cellular functions in genomes of host-adapted pathogens. We found that in the majority of the analyzed genomes, at least some of the genes associated with SSRs encode potential antigens, which is expected if the primary function of SSRs is their contribution to antigenic variation. However, we also found a number of long SSRs associated with housekeeping genes, including rRNA and tRNA genes, genes encoding ribosomal proteins, amino acyl-tRNA synthetases, chaperones, and important metabolic enzymes. Many of these genes are probably essential and it is unlikely that they are phase-variable. Few statistically significant associations between SSRs and gene functional classifications were detected, suggesting that most long SSRs are not related to a particular cellular function or process. Long SSRs in Mycobacterium leprae are mostly associated with pseudogenes and may be contributing to gene loss following the adaptation to an obligate pathogenic lifestyle. We speculate that LSSRs may have played a similar role in genome reduction of other host-adapted pathogens.

Local Renyi entropic profiles of DNA sequences.

BACKGROUND: In a recent report the authors presented a new measure of continuous entropy for DNA sequences, which allows the estimation of their randomness level. The definition therein explored was based on the Rényi entropy of probability density estimation (pdf) using the Parzen's window method and applied to Chaos Game Representation/Universal Sequence Maps (CGR/USM). Subsequent work proposed a fractal pdf kernel as a more exact solution for the iterated map representation. This report extends the concepts of continuous entropy by defining DNA sequence entropic profiles using the new pdf estimations to refine the density estimation of motifs. RESULTS: The new methodology enables two results. On the one hand it shows that the entropic profiles are directly related with the statistical significance of motifs, allowing the study of under and over-representation of segments. On the other hand, by spanning the parameters of the kernel function it is possible to extract important information about the scale of each conserved DNA region. The computational applications, developed in Matlab m-code, the corresponding binary executables and additional material and examples are made publicly available at http://kdbio.inesc-id.pt/~svinga/ep/. CONCLUSION: The ability to detect local conservation from a scale-independent representation of symbolic sequences is particularly relevant for biological applications where conserved motifs occur in multiple, overlapping scales, with significant future applications in the recognition of foreign genomic material and inference of motif structures.

Absence in Helicobacter pylori of an uptake sequence for enhancing uptake of homospecific DNA during transformation.

Uptake sequences are abundant sequence motifs, often located downstream of ORFs, that are used to facilitate the within-species horizontal transfer of DNA. A frequent word analysis of the complete genome sequence of Helicobacter pylori strain 26685 was performed to search for and determine the identity of an uptake sequence in this species. The results demonstrated that Hel. pylori does not possess an uptake sequence. This is the first naturally transformable Gram-negative species shown to lack such a transformation-targeting system.

Microbial gene identification using interpolated Markov models.

This paper describes a new system, GLIMMER, for finding genes in microbial genomes. In a series of tests on Haemophilus influenzae , Helicobacter pylori and other complete microbial genomes, this system has proven to be very accurate at locating virtually all the genes in these sequences, outperforming previous methods. A conservative estimate based on experiments on H.pylori and H. influenzae is that the system finds >97% of all genes. GLIMMER uses interpolated Markov models (IMMs) as a framework for capturing dependencies between nearby nucleotides in a DNA sequence. An IMM-based method makes predictions based on a variable context; i.e., a variable-length oligomer in a DNA sequence. The context used by GLIMMER changes depending on the local composition of the sequence. As a result, GLIMMER is more flexible and more powerful than fixed-order Markov methods, which have previously been the primary content-based technique for finding genes in microbial DNA.

Tn916 transposition in Haemophilus influenzae Rd: preferential insertion into noncoding DNA.

The availability of completely sequenced genomes has created an opportunity for high throughput mutational studies. Using the conjugative transposon Tn916, a pilot project was initiated to determine the efficiency of gene disruption in the first completely sequenced bacterium, Haemophilus influenzae Rd strain KW20. DNA was isolated from Tn916-mutagenized cells, and the point of transposon insertion was determined by inverse PCR, DNA sequencing, and mapping to the wild-type genome sequence. Analysis of the insertion sites at the nucleotide level demonstrated a biased pattern of insertion into regions rich in stretches of A's and T's. Although Tn916 integrated at multiple dispersed positions throughout the chromosome, 9 of 10 insertion events occurred in noncoding, intergenic DNA. It was determined that the intergenic DNA was over 5% more A + T-rich than that of protein coding sequences. This suggests that A + T-rich sequences similar to the Tn916 insertion site would be more likely to reside in the intergenic DNA. In an effort to identify other likely sites for transposon integration, a hidden Markov model of the consensus target insertion site was derived from the Tn916-H. influenzae junction fragments and searched against the entire genome. Eighty percent of the 30 highest-scoring predicted Tn916 target sites were from intergenic, nonprotein-coding regions of the genome. These data support the hypothesis that Tn916 has a marked preference for insertion into noncoding DNA for H. influenzae, suggesting that this mobile element has evolved to minimize disruption of host cell function on integration.

Use of the complete genome sequence information of Haemophilus influenzae strain Rd to investigate lipopolysaccharide biosynthesis.

The availability of the complete 1.83-megabase-pair sequence of the Haemophilus influenzae strain Rd genome has facilitated significant progress in investigating the biology of H.influenzae lipopolysaccharide (LPS), a major virulence determinant of this human pathogen. By searching the H. influenzae genomic database, with sequences of known LPS biosynthetic genes from other organisms, we identified and then cloned 25 candidate LPS genes. Construction of mutant strains and characterization of the LPS by reactivity with monoclonal antibodies, PAGE fractionation patterns and electrospray mass spectrometry comparative analysis have confirmed a potential role in LPS biosynthesis for the majority of these candidate genes. Virulence studies in the infant rat have allowed us to estimate the minimal LPS structure required for intravascular dissemination. This study is one of the first to demonstrate the rapidity, economy and completeness with which novel biological information can be accessed once the complete genome sequence of an organism is available.

Whole-genome random sequencing and assembly of Haemophilus influenzae Rd.

An approach for genome analysis based on sequencing and assembly of unselected pieces of DNA from the whole chromosome has been applied to obtain the complete nucleotide sequence (1,830,137 base pairs) of the genome from the bacterium Haemophilus influenzae Rd. This approach eliminates the need for initial mapping efforts and is therefore applicable to the vast array of microbial species for which genome maps are unavailable. The H. influenzae Rd genome sequence (Genome Sequence DataBase accession number L42023) represents the only complete genome sequence from a free-living organism.