Désirée E. Bennett

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.

PCR-Based Assay for Detection of Neisseria meningitidis Capsular Serogroups 29E, X, and Z

ABSTRACT PCR-based assays for the identification of Neisseria meningitidis serogroups 29E, X, and Z by detection of specific regions of the ctrA gene are described. The specificities of these assays were confirmed using serogroups A, B, C, 29E, H, W135, X, Y, and Z and nongroupable meningococcal isolates.

Consecutive use of two multiplex PCR-based assays for simultaneous identification and determination of capsular status of nine common Neisseria meningitidis serogroups associated with invasive disease.

ABSTRACT We developed two Neisseria meningitidis multiplex PCR assays to be used consecutively that allow determination of the serogroup and capsular status of serogroup A, B, C, 29E, W135, X, and Y cnl-3/cnl-1-like-containing N. meningitidis isolates by direct analysis of the amplicon size. These assays offer a rapid and simple method of serogrouping N. meningitidis.

Development of a diagnostic real-time polymerase chain reaction assay for the detection of invasive Haemophilus influenzae in clinical samples

Since the introduction of the Haemophilus influenzae serotype b vaccine, invasive H. influenzae disease has become dominated by nontypeable (NT) strains. Several widely used molecular diagnostic methods have been shown to lack sensitivity or specificity in the detection of some of these strains. Novel real-time assays targeting the fucK, licA, and ompP2 genes were developed and evaluated. The fucK assay detected all strains of H. influenzae tested (n = 116) and had an analytical sensitivity of 10 genome copies/polymerase chain reaction (PCR). This assay detected both serotype b and NT H. influenzae in 12 previously positive specimens (culture and/or bexA PCR) and also detected H. influenzae in a further 5 of 883 culture-negative blood and cerebrospinal fluid (CSF) samples. The fucK assay has excellent potential as a diagnostic test for detection of typeable and nontypeable strains of invasive H. influenzae in clinical samples of blood and CSF.

Clumping Factor B Promotes Adherence of Staphylococcus aureus to Corneocytes in Atopic Dermatitis

ABSTRACT Staphylococcus aureus skin infection is a frequent and recurrent problem in children with the common inflammatory skin disease atopic dermatitis (AD). S. aureus colonizes the skin of the majority of children with AD and exacerbates the disease. The first step during colonization and infection is bacterial adhesion to the cornified envelope of corneocytes in the outer layer, the stratum corneum. Corneocytes from AD skin are structurally different from corneocytes from normal healthy skin. The objective of this study was to identify bacterial proteins that promote the adherence of S. aureus to AD corneocytes. S. aureus strains from clonal complexes 1 and 8 were more frequently isolated from infected AD skin than from the nasal cavity of healthy children. AD strains had increased ClfB ligand binding activity compared to normal nasal carriage strains. Adherence of single S. aureus bacteria to corneocytes from AD patients ex vivo was studied using atomic force microscopy. Bacteria expressing ClfB recognized ligands distributed over the entire corneocyte surface. The ability of an isogenic ClfB-deficient mutant to adhere to AD corneocytes compared to that of its parent clonal complex 1 clinical strain was greatly reduced. ClfB from clonal complex 1 strains had a slightly higher binding affinity for its ligand than ClfB from strains from other clonal complexes. Our results provide new insights into the first step in the establishment of S. aureus colonization in AD patients. ClfB is a key adhesion molecule for the interaction of S. aureus with AD corneocytes and represents a target for intervention.

Resolution of a Protracted Serogroup B Meningococcal Outbreak with Whole-Genome Sequencing Shows Interspecies Genetic Transfer.

ABSTRACT A carriage study was undertaken (n = 112) to ascertain the prevalence of Neisseria spp. following the eighth case of invasive meningococcal disease in young children (5 to 46 months) and members of a large extended indigenous ethnic minority Traveller family (n = 123), typically associated with high-occupancy living conditions. Nested multilocus sequence typing (MLST) was employed for case specimen extracts. Isolates were genome sequenced and then were assembled de novo and deposited into the Bacterial Isolate Genome Sequencing Database (BIGSdb). This facilitated an expanded MLST approach utilizing large numbers of loci for isolate characterization and discrimination. A rare sequence type, ST-6697, predominated in disease specimens and isolates that were carried (n = 8/14), persisting for at least 44 months, likely driven by the high population density of houses (n = 67/112) and trailers (n = 45/112). Carriage for Neisseria meningitidis (P < 0.05) and Neisseria lactamica (P < 0.002) (2-sided Fishers exact test) was more likely in the smaller, more densely populated trailers. Meningococcal carriage was highest in 24- to 39-year-olds (45%, n = 9/20). Evidence of horizontal gene transfer (HGT) was observed in four individuals cocolonized by Neisseria lactamica and Neisseria meningitidis. One HGT event resulted in the acquisition of 26 consecutive N. lactamica alleles. This study demonstrates how housing density can drive meningococcal transmission and carriage, which likely facilitated the persistence of ST-6697 and prolonged the outbreak. Whole-genome MLST effectively distinguished between highly similar outbreak strain isolates, including those isolated from person-to-person transmission, and also highlighted how a few HGT events can distort the true phylogenetic relationship between highly similar clonal isolates.

PCR and Restriction Endonuclease Assay for Detection of a Novel Mutation Associated with Sulfonamide Resistance in Neisseria meningitidis

ABSTRACT We identified a previously undocumented mutation in the dihydropteroate synthase (folP) gene associated with Neisseria meningitidis sulfonamide resistance. A PCR-based assay to detect this mutation, which is 100% predictive of sulfonamide resistance, was developed.

The widespread use of topical antimicrobials enriches for resistance in Staphylococcus aureus isolated from patients with atopic dermatitis

Carriage rates of Staphylococcus aureus on affected skin in atopic dermatitis (AD) are approximately 70%. Increasing disease severity during flares and overall disease severity correlate with increased burden of S. aureus. Treatment in AD therefore often targets S. aureus with topical and systemic antimicrobials.

来自 AD 患者的金黄色葡萄球菌的局部抗生素耐药性

异位性皮炎(AD, 通常也称为湿疹)是儿童时期最常见的皮肤病之一, 英国和爱尔兰多达 1/4 的儿童受其影响。AD 患者经常在其皮肤上携带金黄色葡萄球菌。当患有 AD 的个体出现爆发时,即皮肤发红和瘙痒增加,皮肤上通常会有更多的金黄色葡萄球菌。因此通常用抗生素治疗 AD。这项研究在爱尔兰都柏林和英国圣安德鲁斯进行,旨在了解与未患 AD 的儿童相比, AD 患儿的金黄色葡萄球菌的抗生素耐药性(意味着细菌能够抵抗杀死它们的抗生素)的差异。从 50 名在皮肤病诊所就诊的患有 AD 的儿童中收集皮肤拭子,并在急诊科就诊的 49 名儿童中收集拭子,这些儿童在其鼻腔中无害地携带细菌。检查来自两组儿童的金黄色葡萄球菌的DNA(遗传密码),并用常用抗生素测试样品以鉴定哪些抗生素成功地杀死细菌。来自 AD 儿童的总体样本对抗生素夫西地酸 (FA) 的耐药性是未患 AD 儿童的两倍多,该药物通常以乳膏形式治疗皮肤感染。遗传密码显示,与未患 AD 的儿童相比,AD 患儿的样本中通过不同机制发生了对 FA 的耐药性。最后,可能允许细菌在抗菌皂中存活的基因在 AD 样本中比在非 AD 样本中常见八倍。这表明用于 AD 的治疗可以影响来自 AD 患者的金黄色葡萄球菌对抗生素的耐药性。

Topical antimicrobial resistance in S. aureus from patients with AD

Atopic dermatitis (AD, commonly also known as eczema) is one of the commonest skin diseases of childhood, with up to 1 in 4 children in the U.K. and Ireland affected. People with AD very frequently carry the bacterium Staphylococcus aureus on their skin. When individuals with AD have a flare, with increased redness and itchiness of their skin, there usually is more S. aureus on the skin. Antibiotics are therefore commonly used as a treatment in AD. This study, carried out in Dublin, Ireland and St Andrews, U.K., aimed to find out if there were differences in antibiotic resistance (meaning the bacteria are able to resist to the antibiotics meant to kill them) in S. aureus from children with AD, compared with children who do not have AD. Skin swabs were collected from 50 children with AD attending a dermatology clinic and swabs from 49 children being seen at an Emergency Department, harmlessly carrying the bacteria in their nose. The DNA (genetic code) of the S. aureus from both groups of children was examined, and samples tested with common antibiotics to identify which ones worked to kill the bacteria. Overall samples from children with AD were more than twice as frequently resistant to the antibiotic fusidic acid (FA), often used in a cream to treat skin infections. The genetic code revealed that resistance to FA happens by different mechanisms in samples from children with AD compared to children without AD. Finally, genes that might allow bacteria to survive against antiseptic soaps were eight times commoner in AD samples. This shows that the treatments used for AD can influence resistance to antibiotics in S. aureus from people with AD.