Mathew Diggle

Interlaboratory Comparison of PCR-Based Identification and Genogrouping of Neisseria meningitidis

ABSTRACT Twenty clinical samples (18 cerebrospinal fluid samples and 2 articular fluid samples) were sent to 11 meningococcus reference centers located in 11 different countries. Ten of these laboratories are participating in the EU-MenNet program (a European Union-funded program) and are members of the European Monitoring Group on Meningococci. The remaining laboratory was located in Burkina Faso. Neisseria meningitidis was sought by detecting several meningococcus-specific genes (crgA, ctrA, 16S rRNA, and porA). The PCR-based nonculture method for the detection of N. meningitidis gave similar results between participants with a mean sensitivity and specificity of 89.7 and 92.7%, respectively. Most of the laboratories also performed genogrouping assays (siaD and mynB/sacC). The performance of genogrouping was more variable between laboratories, with a mean sensitivity of 72.7%. Genogroup B gave the best correlation between participants, as all laboratories routinely perform this PCR. The results for genogroups A and W135 were less similar between the eight participating laboratories that performed these PCRs.

Target Gene Sequencing To Characterize the Penicillin G Susceptibility of Neisseria meningitidis

ABSTRACT Clinical isolates of Neisseria meningitidis with reduced susceptibility to penicillin G (intermediate isolates, PenI) harbor alterations in the penA gene encoding the penicillin binding protein 2 (PBP2). A 402-bp DNA fragment in the 3′ half of penA was sequenced from a collection of 1,670 meningococcal clinical isolates from 22 countries that spanned 60 years. Phenotyping, genotyping, and the determination of MICs of penicillin G were also performed. A total of 139 different penA alleles were detected with 38 alleles that were highly related, clustered together in maximum-likelihood analysis and corresponded to the penicillin G-susceptible isolates. The remaining 101 penA alleles were highly diverse, corresponded to different genotypes or phenotypes, and accounted for 38% of isolates, but no clonal expansion was detected. Analysis of the altered alleles that were represented by at least five isolates showed high correlation with the PenI phenotype. The deduced amino acid sequence of the corresponding PBP2 comprised five amino acid residues that were always altered. This correlation was not complete for rare alleles, suggesting that other mechanisms may also be involved in conferring reduced susceptibility to penicillin. Evidence of mosaic structures through events of interspecies recombination was also detected in altered alleles. A new website was created based on the data from this work (http://neisseria.org/nm/typing/penA ). These data argue for the use of penA sequencing to identify isolates with reduced susceptibility to penicillin G and as a tool to improve typing of meningococcal isolates, as well as to analyze DNA exchange among Neisseria species.

Comparison of Commercial DNA Extraction Kits for Extraction of Bacterial Genomic DNA from Whole-Blood Samples

ABSTRACT The demand for molecular diagnostic tests in medical microbiology has highlighted the need for efficient methods of DNA extraction. In addition, it is preferable for these methods to be automated. An example of such a requirement is for the confirmation of meningococcal disease where rapid, sensitive, and specific procedures are required for public health management purposes. Previous studies have shown that whole blood is the preferred method for the isolation of bacterial DNA in meningococcal disease, and in this study, we compare five commercially available kits for the extraction of bacterial genomic DNA from whole-blood samples. These include kits in a 96-well binding plate, 96-well filter plate, and metallic bead formats. The method for all five kits is described, and the sensitivity, specificity, ease of automation, and overall efficiency are determined.

Analysis of genetic relatedness of Haemophilus influenzae isolates by multilocus sequence typing.

The gram-negative bacterium Haemophilus influenzae is a human-restricted commensal of the nasopharynx that can also be associated with disease. The majority of H. influenzae respiratory isolates lack the genes for capsule production and are nontypeable (NTHI). Whereas encapsulated strains are known to belong to serotype-specific phylogenetic groups, the structure of the NTHI population has not been previously described. A total of 656 H. influenzae strains, including 322 NTHI strains, have been typed by multilocus sequence typing and found to have 359 sequence types (ST). We performed maximum-parsimony analysis of the 359 sequences and calculated the majority-rule consensus of 4,545 resulting equally most parsimonious trees. Eleven clades were identified, consisting of six or more ST on a branch that was present in 100% of trees. Two additional clades were defined by branches present in 91% and 82% of trees, respectively. Of these 13 clades, 8 consisted predominantly of NTHI strains, three were serotype specific, and 2 contained distinct NTHI-specific and serotype-specific clusters of strains. Sixty percent of NTHI strains have ST within one of the 13 clades, and eBURST analysis identified an additional phylogenetic group that contained 20% of NTHI strains. There was concordant clustering of certain metabolic reactions and putative virulence loci but not of disease source or geographic origin. We conclude that well-defined phylogenetic groups of NTHI strains exist and that these groups differ in genetic content. These observations will provide a framework for further study of the effect of genetic diversity on the interaction of NTHI with the host.

Semiautomation of Multilocus Sequence Typing for the Characterization of Clinical Isolates of Neisseria meningitidis

ABSTRACT The Scottish Meningococcus and Pneumococcus Reference Laboratory (SMPRL) provides a national service for the laboratory confirmation of meningococcal and pneumococcal disease in Scotland. Part of this service includes the serogrouping of meningococcal isolates followed by typing and subtyping. The procedures for this are labor-intensive but important for the identification of linked cases and the surveillance of disease so that effective public health measures can be taken. However, different strains of meningococci, such as those within the electrophoretic type 37 complex, occurring during case clusters of disease are now indistinguishable by current methods. The SMPRL has started using multilocus sequence typing (MLST) as a routine method for the characterization of isolates of Neisseria meningitidis. MLST produces nucleotide sequence data of seven housekeeping genes providing results that are useful for public health management. However, the method is laborious and time-consuming and therefore lends itself towards automation. The SMPRL therefore developed a semiautomated method for MLST using a 96-well format liquid handler and an automated DNA sequencer. Semiautomated MLST is now provided as a reference service for Scotland. This work describes the methodology required for the characterization of N. meningitidis and highlights its usefulness for public health intervention.

Automated pneumococcal MLST using liquid-handling robotics and a capillary DNA sequencer

Multilocus sequence typing (MLST) is used by the Scottish Meningococcus and Pneumococcus Reference Laboratory (SMPRL) as a routine method for the characterization of certain bacterial pathogens. The SMPRL recently started performing MLST on strains of Streptococcus pneumoniae, and here we describe a fully automated method for MLST using a 96-well-format liquid-handling robot and a 96-capillary automated DNA sequencer.

Increased Genetic Diversity of Neisseria meningitidis Isolates after the Introduction of Meningococcal Serogroup C Polysaccharide Conjugate Vaccines

ABSTRACT During the 1990s, the incidence of meningococcal disease was high in the United Kingdom. This was due primarily to an increase in serogroup C disease, particularly that within the ET-37/ST-11 genetic lineage. Serogroup C meningococcal polysaccharide conjugate vaccines were introduced in the United Kingdom in 1999, but the sequence types of meningococci causing disease since that time have not yet been reported. We have used serogrouping and multilocus sequence typing to characterize meningococci from patients with invasive disease over a 4-year period and show that there is a significant increase in genetic diversity but no genetic evidence of capsule switching.

Analysis of PorA variable region 3 in meningococci: implications for vaccine policy?

Outer membrane protein (OMP) vaccines are being developed against Neisseria meningitidis serogroup B which may provide protection against common circulating serotypes and serosubtypes in some countries. However, limited data is available in Europe from genosubtyping meningococci. We therefore undertook a retrospective analysis of the three main variable regions, VR1, VR2 as well as VR3, of the porA gene from N. meningitidis isolated from different countries, mainly from Scotland and Sweden. Analysis of this gene showed that, amongst 226 strains studied, there were a total of 78 different strains. No new VR1 or VR2 alleles were found but five new VR3 alleles are described. Our data indicates the importance of analysing the VR3 region of PorA in addition to VR1 and VR2 and also highlights, in general terms, the need for genosubtyping meningococci. Such analyses have major implications for the design of new meningococcal vaccines.

Multidrug-Resistant Escherichia coli Bacteremia

To the Editor: Extraintestinal pathogenic Escherichai coli (ExPEC) bacteria have the ability to cause diverse and serious diseases, such as urinary tract infections (UTIs) and bacteremia (1–3); incidence of bacteremia is increasing globally (4). The emergence of multidrug resistance in E. coli is also becoming a global concern, with particular emphasis on E. coli sequence type (ST) 131, which is being increasingly reported in UTIs. Drug resistance is mediated by extended-spectrum β-lactamases (ESBLs), mainly of the CTX-M family, particularly CTX-M-15 and 14, and less frequently of the SHV and OXA families (5,6). Few studies are available regarding the characterization of E. coli strains causing bacteremia. We characterized 140 E. coli isolates from bacteremia patients treated at Nottingham University Hospital (Nottingham, UK) over a 5-month period, with the aim of developing an epidemiologic profile of the population of ExPEC that causes bacteremia. For context, we compared the isolates with 125 E. coli isolates from urine samples collected during the same period. Cases were selected to include isolates from a diverse patient group: patient ages ranged from 1 month to 90 years; patient sex was evenly divided between male and female; infections were community- and hospital-associated; and suspected sources of infection varied. Antimicrobial drug susceptibility tests, PCR detection of ESBL genes, multilocus sequence typing using the Achtman scheme (http://mlst.ucc.ie/mlst/dbs/Ecoli), and virulence-associated gene (VAG) carriage screening by PCR were performed on isolates as described (7). Significantly more bacteremia E. coli isolates than urine E. coli isolates were resistant to ciprofloxacin (25.7% vs. 8.8%; p 2 classes); a significantly higher number of multidrug-resistant bacteremia E. coli isolates than multidrug-resistant urine isolates were found (50.7% vs. 32%; p = 0.01). PCR screening for ESBL carriage showed significantly higher ESBL carriage in bacteremia E. coli isolates than urine isolates for blaSHV (15.7% vs. 5.6%; p = 0.008), blaCTX-M (29.3% vs. 17.6%; p = 0.025), and blaOXA (14.3% vs. 6.4%; p = 0.037). Total ESBL carriage for bacteremia isolates was also significantly higher than for urine isolates (59.3% vs. 29.6%; p<0.001). Multilocus sequence types were determined for all E. coli isolates. A total of 63 STs were found among the urine isolates (Figure, panel A); the highest prevalence was ST73 (n = 16, 12.8%), followed by ST131 (n = 9, 7.2%), ST69 (n = 9, 7.2%), ST95 (n = 6, 4.8%), ST404 (n = 6, 4.8%), ST127 (n = 4, 3.2%), ST141 (n = 4, 3.2%), and ST10 (n = 3, 2.4%). Prevalence patterns of STs among bacteremia E. coli isolates were noticeably different (Figure, panel B). Three main STs were obtained. ST131 dominated (n = 30, 21.43%) and was significantly higher in prevalence than for the urine isolates (p<0.001). ST73 (n = 24, 17.14%) and ST95 (n = 13, 9.29%) were the other 2 primary STs found. The 8 most prevalent STs in the bacteremia isolates represented 59.29% of the total population, whereas the 8 most prevalent STs in the urine isolates represented 45.6% of the total population. This finding is suggestive of selection of a smaller number of dominant STs in bacteremia. Figure Minimum-spanning trees showing carriage of extended-spectrum β-lactamases (ESBL) in Escherichia coli isolates from urine samples (A) and samples from patients with bacteremia (B). Each circle represents 1 sequence type (ST), and the size of the ... ESBL carriage was mapped onto minimum-spanning trees for the 2 isolate groups. ESBL carriage among urine isolates was focused on a small number of STs; 19 (30.16%) of the 63 STs contained ESBL-positive isolates (Figure, panel A). The predominant ST73 group contained 18.75% ESBL-positive isolates; the other predominant STs exhibited ESBL-positive isolates at the following levels: ST131 (44.44%), ST69 (33.33%), ST95 (50%), and ST10 (0%). In contrast, 30 (51.72%) of the 58 STs among bacteremia isolates contained ESBL-positive isolates, significantly higher than for the urine isolates (p = 0.016). At the ST level, predominant STs had higher ESBL carriage in the bacteremia isolates than in the urine isolates: ST131 (50%), ST73 (50%), ST12 (75%), ST10 (100%), ST14 (50%), ST2278 (33.33%). ST95 (46.15%) and ST69 (20%) showed comparable levels. These results suggest that ESBL drug resistance is selecting for dominant ExPEC bacteremia strains. To investigate whether the differences in ST observations between bacteremia and urine isolates could be attributable to differences in virulence genes, VAGs of all isolates were screened by multiplex PCR. VAGs were found equally distributed across the 2 populations, with no statistically significant difference (p = 0.675). Comparison of serum resistance levels between urine and blood isolates also showed no phenotypic differences. In conclusion, we found high levels of ESBL carriage and multidrug resistance in ExPEC isolates that cause bacteremia. A comparison with urine isolates provided evidence that ESBL-mediated drug resistance appears to be the selective pressure in the emergence of dominant STs in bacteremia. Future research should focus on identifying if prolonged antimicrobial drug treatment in bacteremia patients is leading to this selection.

Molecular methods for the detection and characterization of Neisseria meningitidis

Neisseria meningitidis remains a common global cause of morbidity and mortality. The laboratory confirmation of meningococcal disease is, therefore, very important for individual patient management and for public health management. Through surveillance schemes, it provides long-term epidemiologic data that can be used to inform vaccine policy. Traditional methods, such as latex agglutination and the enzyme-linked immunosorbent assay, are still used, but molecular methods are now also established. In this review, molecular methods for the laboratory confirmation and characterization of meningococci are described. PCR is an invaluable tool in modern biology and can be used to predict the group, type and subtype of meningococci. It is now also used in a fluorescence-based format for increased sensitivity and specificity. The method also provides the amplified DNA for other techniques, such as multilocus sequence typing. Other methods for the discrimination of meningococci have also played and continue to play an important part in epidemiology. For example, pulsed-field gel electrophoresis is highly discriminatory, whilst multilocus enzyme electrophoresis provided the basis for the description of global meningococcal clones and formed the foundation for multilocus sequence typing. Other less commonly used methods, such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and pyrosequencing, may increasingly find their way into microbiology reference laboratories. Nevertheless, nucleotide sequencing and laboratory automation have aided the introduction of many methods and provide data that are digitally based and, therefore, highly accurate and portable.