Timothy G. Harrison

Consensus Sequence-Based Scheme for Epidemiological Typing of Clinical and Environmental Isolates of Legionella pneumophila

ABSTRACT A previously described sequence-based epidemiological typing method for clinical and environmental isolates of Legionella pneumophila serogroup 1 was extended by the investigation of three additional gene targets and modification of one of the previous targets. Excellent typeability, reproducibility, and epidemiological concordance were determined for isolates belonging to both serogroup 1 and the other serogroups investigated. Gene fragments were amplified from genomic DNA, and PCR amplicons were sequenced by using forward and reverse primers. Consensus sequences are entered into an online database, which allows the assignment of individual allele numbers. The resulting sequence-based type or allelic profile comprises a string of the individual allele numbers separated by commas, e.g., 1,4,3,1,1,1, in a predetermined order, i.e., flaA, pilE, asd, mip, mompS, and proA. The index of discrimination (D) obtained with these six loci was calculated following analysis of a panel of 79 unrelated clinical isolates. A D value of >0.94 was obtained, and this value appears to be sufficient for use in the epidemiological investigation of outbreaks caused by L. pneumophila. The D value rose to 0.98 when the results of the analysis were combined with those of monoclonal antibody subgrouping. Sequence-based typing of L. pneumophila is epidemiologically concordant and discriminatory, and the data are easily transportable. This consensus method will assist in the epidemiological investigation of L. pneumophila infections, especially travel-associated cases, by which it will allow a rapid comparison of isolates obtained in more than one country.

Proposals to unify the genera Grahamella and Bartonella, with descriptions of Bartonella talpae comb. nov., Bartonella peromysci comb. nov., and three new species, Bartonella grahamii sp. nov., Bartonella taylorii sp. nov., and Bartonella doshiae sp. nov.

Polyphasic methods were used to examine the taxonomic positions of three newly identified Grahamella species. A comparison of the 16S rRNA gene sequences of these organisms with the sequences available for other bacteria revealed that these three species form a tight monophyletic cluster with members of the genus Bartonella. This cluster is only remotely related to other members of the order Rickettsiales. Determinations of the levels of DNA relatedness between Grahamella species and Bartonella species (by using a modified hydroxyapatite method) revealed that all of the species belonging to these two genera are distinct but closely related. On the basis of these data and the results of guanine-plus-cytosine content and phenotypic characterization studies, we propose that the genera Grahamella and Bartonella should be unified and that the latter name should be retained. Bartonella talpae and Bartonella peromysci, new combinations for former Grahamella species, are created, and the following three new Bartonella species are described: Bartonella grahamii, Bartonella taylorii, and Bartonella doshiae. A taxonomic analysis of Grahamella species complete the study of all members of the family Bartonellaceae, and the results of this study support the proposal that the family should be transferred out of the order Rickettsiales.

Distribution of Legionella pneumophila serogroups, monoclonal antibody subgroups and DNA sequence types in recent clinical and environmental isolates from England and Wales (2000-2008)

Clinical isolates of Legionella pneumophila, obtained from 167 patients, who acquired their illness in the community in England and Wales between January 2000 and March 2008, were compared with 276 environmental isolates of L. pneumophila obtained over the same period as part of the routine sampling of ‘managed’ water systems. The 443 isolates were typed by monoclonal antibody (mAb) subgrouping and the internationally standardised, seven-gene loci, sequence-based typing (SBT) scheme of the European Working Group for Legionella Infections (EWGLI). Of the clinical isolates, 97.6% were L. pneumophila serogroup (sgp) 1, compared with only 55.8% of environmental isolates (P = 0.0002); 91.6% were subgrouped as mAb3/1+ve, compared with only 8.3% of environmental isolates (P < 0.0001). The isolates were very diverse, with SBT identifying 111 sequence types (STs) (index of diversity [IOD] 0.954). Among the clinical isolates, 42 ST were seen, with one (ST47) accounting for 25.7% and three (ST47, ST37 and ST62) accounting for 46.1% of all isolates. Eighty-two STs were identified among the environmental isolates, with two (ST1 and ST79) accounting for 34.1% of these. Comparison of the STs seen among clinical and environmental isolates showed that there was very little overlap between the two populations (P < 0.0001), with common clinical strains found in the environment very infrequently: 0.4, 0.7 and 0% (ST47, ST37 and ST62, respectively), and common environmental strains rarely causing disease: 4.8 and 1.2% (ST1 and ST79, respectively). Combining phenotypic and genotypic data identified 144 phenons (IOD 0.970); 52 among clinical isolates and 101 among environmental isolates. The most abundant clinical strain, mAb ‘Allentown’ ST47, accounted for 22.8% of cases, but was only found once in the environment. Conversely, mAb ‘Oxford/OLDA’ ST1 was the most common environmental strain (17.0%), but only caused two infections. A review of the published data shows that mAb ‘Allentown’ ST47 is also an important cause of infection in France and possibly in the Netherlands. However, it was not found in a large study of German clinical isolates. This study confirms previous work showing that just a few strains of L. pneumophila cause the majority of community-acquired Legionella infection in England and Wales, and that these clinically significant strains are only rarely found in managed water systems. These data suggest that knowing which particular strain is present in an environment might be at least as important as knowing the quantity in which legionellae are present.

Grahamella in small woodland mammals in the U.K.: isolation, prevalence and host specificity

Bacteria isolated from the blood of small woodland mammals were identified as members of the genus Grahamella. The prevalence of Grahamella infection among the 37 small mammals examined, detected by cultivation of blood samples, was 62%. This figure is somewhat higher than previous reports. Further characterization of the isolates, based on restriction enzyme analysis of the 16S rRNA gene, serological reactivity and DNA hybridization studies, revealed three distinct Grahamella species. One of the species was found in five different species of small mammal (Apodemus sylvaticus, A. flavicollis, Clethrionomys glareolus, Microtus agrestis and Neomys fodiens). All three species were found in M. agrestis, although there was no evidence of concurrent infection of an animal by more than one species of Grahamella. These observations demonstrate that Grahamella spp. are not host-specific, as previously thought, and that it is therefore invalid to name Grahamella spp. solely on the basis of the host in which they are observed.

Legionella pneumophila Strain 130b Possesses a Unique Combination of Type IV Secretion Systems and Novel Dot/Icm Secretion System Effector Proteins

Legionella pneumophila is a ubiquitous inhabitant of environmental water reservoirs. The bacteria infect a wide variety of protozoa and, after accidental inhalation, human alveolar macrophages, which can lead to severe pneumonia. The capability to thrive in phagocytic hosts is dependent on the Dot/Icm type IV secretion system (T4SS), which translocates multiple effector proteins into the host cell. In this study, we determined the draft genome sequence of L. pneumophila strain 130b (Wadsworth). We found that the 130b genome encodes a unique set of T4SSs, namely, the Dot/Icm T4SS, a Trb-1-like T4SS, and two Lvh T4SS gene clusters. Sequence analysis substantiated that a core set of 107 Dot/Icm T4SS effectors was conserved among the sequenced L. pneumophila strains Philadelphia-1, Lens, Paris, Corby, Alcoy, and 130b. We also identified new effector candidates and validated the translocation of 10 novel Dot/Icm T4SS effectors that are not present in L. pneumophila strain Philadelphia-1. We examined the prevalence of the new effector genes among 87 environmental and clinical L. pneumophila isolates. Five of the new effectors were identified in 34 to 62% of the isolates, while less than 15% of the strains tested positive for the other five genes. Collectively, our data show that the core set of conserved Dot/Icm T4SS effector proteins is supplemented by a variable repertoire of accessory effectors that may partly account for differences in the virulences and prevalences of particular L. pneumophila strains.

Sequence-Based Typing of Legionella pneumophila Serogroup 1 Offers the Potential for True Portability in Legionellosis Outbreak Investigation

ABSTRACT Seven gene loci of Legionella pneumophila serogroup 1 were analyzed as potential epidemiological typing markers to aid in the investigation of legionella outbreaks. The genes chosen included four likely to be selectively neutral (acn, groES, groEL, and recA) and three likely to be under selective pressure (flaA, mompS, and proA). Oligonucleotide primers were designed to amplify 279- to 763-bp fragments from each gene. Initial sequence analysis of the seven loci from 10 well-characterized isolates of L. pneumophila serogroup 1 gave excellent reproducibility (R) and epidemiological concordance (E) values (R = 1.00; E = 1.00). The three loci showing greatest discrimination and nucleotide variation, flaA, mompS, and proA, were chosen for further study. Indices of discrimination (D) were calculated using a panel of 79 unrelated isolates. Single loci gave D values ranging from 0.767 to 0.857, and a combination of all three loci resulted in a D value of 0.924. When all three loci were combined with monoclonal antibody subgrouping, the D value was 0.971. Sequence-based typing of L. pneumophila serogroup 1 using only three loci is epidemiologically concordant and highly discriminatory and has the potential to become the new “gold standard” for the epidemiological typing of L. pneumophila.

Pneumococcal carriage in children and adults two years after introduction of the thirteen valent pneumococcal conjugate vaccine in England.

BACKGROUND/AIMS In April 2010 the 7-valent pneumococcal conjugate vaccine (PCV7) was replaced by the 13-valent PCV. We investigated pneumococcal carriage in children eligible for PCV7 or PCV13 and their household contacts. METHODS Eligible families in Hertfordshire and Gloucester were identified and a nasopharyngeal swab obtained from consenting household members between July 2012 and March 2013. Samples were cultured for Streptococcus pneumoniae and serotyped by standard methods. For each serotype the ratio of its prevalence in invasive pneumococcal disease (IPD) to its carriage prevalence (case:carrier ratio, CCR) was calculated. Results were compared with previous carriage studies in 2001/2002 and 2008/2009, before and after PCV7 introduction. RESULTS 217 households were included. Among <5-year olds 47.7% (95% confidence interval 41.8-53.5) were carrying a pneumococcus compared with 51.0% (95% CI: 44.0-58.0) in 2008/2009 and 48.4% (95% CI: 44.1-52.7) in 2001/2002. The odds of carrying a PCV7 serotype was significantly reduced in 2008/2009 (0.07, 95% CI: 0.03-0.16) and 2012/2013 (0.01 95% CI: 0.00-0.07) relative to 2001/2002, while the odds of carrying any of the extra six PCV13 serotypes increased after PCV7 introduction (1.38, 95%CI: 0.73-2.59) but declined significantly after PCV13 introduction (0.05, 95%CI: 0.01-0.37). The CCRs for the frequently carried serotypes were relatively low, with the highest CCR observed for serotypes 7F, 19A, 3, 8, and 33F. Across the three carriage studies, CCR estimates were stable for nearly all serotypes. CONCLUSION Carriage of additional PCV13 serotypes has rapidly reduced post-PCV13 introduction in both vaccinated and unvaccinated individuals with a continued decline in transmission of PCV7 serotypes. Carriage rates in children remain unchanged, but the low CCRs of replacing serotypes would be expected to further reduce overall IPD across all age groups.

Whooping cough in school age children with persistent cough: prospective cohort study in primary care

Abstract Objective To estimate the proportion of school age children with a persistent cough who have evidence of a recent Bordetella pertussis infection. Design Prospective cohort study (October 2001 to March 2005). Setting General practices in Oxfordshire, England. Participants 172 children aged 5-16 years who presented to their general practitioner with a cough lasting 14 days or more who consented to have a blood test. Main outcome measures Serological evidence of a recent Bordetella pertussis infection; symptoms at presentation; duration and severity of cough; sleep disturbance (parents and child). Results 64 (37.2%, 95% confidence interval 30.0% to 44.4%) children had serological evidence of a recent Bordetella pertussis infection; 55 (85.9%) of these children had been fully immunised. At presentation, children with whooping cough were more likely than others to have whooping (odds ratio 2.85, 95% confidence interval 1.39 to 5.82), vomiting (4.35, 2.04 to 9.25), and sputum production (2.39, 1.14 to 5.02). Children with whooping cough were also more likely to still be coughing two months after the start of their illness (85% v 48%; P = 0.001), continue to have more than five coughing episodes a day (P = 0.049), and cause sleep disturbance for their parents (P = 0.003). Conclusions For school age children presenting to primary care with a cough lasting two weeks or more, a diagnosis of whooping cough should be considered even if the child has been immunised. Making a secure diagnosis of whooping cough may prevent inappropriate investigations and treatment.

Genotypic Variation in the Bordetella pertussis Virulence Factors Pertactin and Pertussis Toxin in Historical and Recent Clinical Isolates in the United Kingdom

ABSTRACT The reemergence of pertussis has been reported in several countries despite high vaccination coverage. Studies in The Netherlands and Finland have investigated polymorphism in the genes coding for two important virulence factors of Bordetella pertussis, pertactin and pertussis toxin, and identified the emergence and subsequent dominance in circulating strains of pertactin and toxin variants not found in the whole-cell vaccine (WCV). The study described here investigated whether such variation had occurred in the United Kingdom, which presently has low levels of pertussis. Sequence analysis of the genes for pertactin (prnA) and the pertussis toxin S1 subunit (ptxA) among isolates of B. pertussis from 285 United Kingdom patients, from 1920 to 1999, revealed three prnA variants, prnA(1),prnA(2), and prnA(3), and twoptxA variants, ptxA(1) andptxA(2), showing differences in nucleic acid sequence. The proportion of pertactin gene types not included in the United Kingdom WCV, i.e., prnA(2) and prnA(3), has increased in recent years and was found in 21 of 86 (24%) strains from the 1980s and 56 of 105 (53%) strains from the 1990s. To date, the presence of these nonvaccine prnA types has not been associated with a resurgence of pertussis in the United Kingdom. The distribution of prnA andptxA types in The Netherlands, Finland, and the United Kingdom in the 1990s is distinct. The most striking difference in the United Kingdom isolates is that all 105 of the most recent circulating strains (from 1998 to 1999) are of a pertussis toxin type found in the United Kingdom WCV, i.e., ptxA(1).

Diagnosis of Streptococcus pneumoniae Infections in Adults with Bacteremia and Community-Acquired Pneumonia: Clinical Comparison of Pneumococcal PCR and Urinary Antigen Detection

ABSTRACT The diagnosis of severe Streptococcus pneumoniae infection relies heavily on insensitive culture techniques. To improve the usefulness of PCR assays, we developed a dual-PCR protocol (targeted at pneumolysin and autolysin) for EDTA blood samples. This was compared to the Binax NOW S. pneumoniae urine antigen test in patients with bacteremic pneumococcal infections. Patients with nonbacteremic community-acquired pneumonia also were tested by these methods to determine what proportion could be confirmed as pneumococcal infections. A direct comparison was made in a group of patients who each had both tests performed. The Binax NOW S. pneumoniae urine antigen test was positive in 51 of 58 bacteremic pneumococcal cases (sensitivity, 88%; 95% confidence interval [CI], 77 to 95%), whereas the dual PCR was positive in 31 cases (sensitivity, 53.5%; 95% CI, 40 to 67%; P < 0.0001), and all of these had detectable urinary antigens. Both tests gave positive results in 2 of 51 control patients (referred to as other-organism septicemia), giving a specificity of 96% (95% CI, 86.5 to 99.5%). In 77 patients with nonbacteremic community-acquired pneumonia, urinary antigen was detected significantly more often (in 21 patients [27%]) than a positive result by the dual-PCR protocol (6 [8%]) (P = 0.002). The development of a dual-PCR protocol enhanced the sensitivity compared to that of the individual assays, but it is still significantly less sensitive than the Binax NOW urine antigen test, as well as being more time-consuming and expensive. Urinary antigen detection is the nonculture diagnostic method of choice for patients with possible severe pneumococcal infection.