Margareta Granlund

Multilocus Sequence Typing of Swedish Invasive Group B Streptococcus Isolates Indicates a Neonatally Associated Genetic Lineage and Capsule Switching

ABSTRACT Streptococcus agalactiae, also designated group B streptococcus (GBS), is an important pathogen in neonates, pregnant women, and nonpregnant adults with predisposing conditions. We used multilocus sequence typing (MLST) to characterize 158 GBS isolates that were associated with neonatal and adult invasive disease and that were collected in northern and western Sweden from 1988 to 1997. Five major genetic lineages (sequence type [ST] 19, ST-17, ST-1, ST-23, and ST-9 complexes) were identified among the isolates, including serotype Ia, Ib, and II to V isolates, indicating a highly clonal population structure among invasive GBS isolates. A number of STs were found to contain isolates of different serotypes, which indicates that capsule switching occurred rather frequently. Two distantly related genetic lineages were identified among isolates of serotype III, namely, clonal complex 19 (CC19), and CC17. CC19 was equally common among isolates from adult and neonatal disease (accounting for 10.3% of GBS isolates from adult disease and 18.7% from neonatal disease), whereas CC17 significantly appeared to be associated with neonatal invasive disease (isolated from 21.9% of neonatal isolates but only 2.6% of adult isolates). The distribution of the mobile elements GBSi1 and IS1548 reveals that they can act as genetic markers for lineages CC17 and CC19, respectively.

Mutually Exclusive Distribution of IS1548 and GBSi1, an Active Group II Intron Identified in Human Isolates of Group B Streptococci

The present study shows that active, self-splicing group II intron GBSi1 is located downstream of the C5a-peptidase gene, scpB, in some group B streptococcus (GBS) isolates that lack insertion sequence IS1548. IS1548 was previously reported to be often present at the scpB locus in GBS isolated in association with endocarditis. Since none of 67 GBS isolates examined, 40 of which were of serotype III, harbored both IS1548 and GBSi1, these two elements are suggested to be markers for different genetic lineages in GBS serotype III. The DNA region downstream of scpB in GBS isolates harboring either GBSi1, IS1548, or none of these mobile elements was found to encode the laminin binding protein, Lmb, which shows sequence similarities to a family of streptococcal adhesins. IS1548 is inserted 9 bp upstream of the putative promoter for lmb, while the insertion site for GBSi1 is located 88 bp further upstream. Sequences highly similar to GBSi1 exist also in Streptococcus pneumoniae. An inverted repeat sequence, with features typical of transcription terminators, was identified immediately upstream of the insertion site for the group II intron both in the GBS and S. pneumoniae sequences. This motif is suggested to constitute a target for the GBS intron as well as for rather closely related introns in Bacillus halodurans, Pseudomonas alcaligenes, and Pseudomonas putida. When transcripts containing the GBSi1 intron were incubated at high concentrations of ammonium and magnesium, a major product with the expected length and sequence for the ligated exons was generated. Unlike, however, all members of group II investigated so far, the excised intron was in linear, rather than in a branched (lariat), form.

Identification of a Novel Insertion Element, IS1548, in Group B Streptococci, Predominantly in Strains Causing Endocarditis

Hyaluronidase has been postulated to be a virulence factor in group B streptococci (GBS). No hyaluronidase activity was found in 15 of 50 GBS isolates from adults studied. Most of these hyaluronidase-negative strains belonged to serotype III. In strains lacking hyaluronidase activity, an insertion of 1317 nucleotides was found in the hyaluronidase gene. The fragment was cloned and sequenced and found to have characteristics of a novel insertion sequence, designated IS1548. As well as in GBS serotype III, this sequence was found in 3 of 6 serotype II isolates and in all 10 group A streptococcal strains (GAS) tested. Homologies were found with repeated sequences in Streptococcus pneumoniae and with H repeats in Escherichia coli. All GBS strains harboring IS1548 and some GAS strains had one copy of IS1548 located downstream of the C5a peptidase gene. IS1548 was present in 9 of 13 GBS isolates from blood in endocarditis patients and in 3 of 22 vaginally colonizing strains.

Subtype analysis of Blastocystis isolates in Swedish patients.

Blastocystis is a genetically diverse and widespread intestinal parasite of animals and humans with controversial pathogenic potential. At least nine subtypes of Blastocystis have been found in humans. The genetic diversity of Blastocystis was examined in stool samples from 68 patients from the Stockholm area, Sweden. Blastocystis was identified by light microscopy, and subtyped by sequencing the 5′-end of the small subunit ribosomal RNA gene. Five Blastocystis subtypes were identified in the 63 patients whose samples were successfully subtyped: ST1 (15.9%), ST2 (14.3%), ST3 (47.6%), ST4 (20.6%), and ST7 (1.6%). ST3 was more common in males compared to females (P = 0.049). Comparative molecular analysis of Blastocystis sequences revealed intra-subtype variations within the identified subtypes with the exception of ST4. Among ST4 sequences in this study, as well as in the majority of human GenBank sequences, a limited genetic diversity was found compared to what was found among the other common subtypes (ST1, ST2 and ST3). The relative prevalence of ST4 in this study was comparable to the overall distribution of ST4 in European cohorts (16.5%). This contrasts with the sparse reports of ST4 in studies from other continents, which may indicate that the distribution of this subtype is geographically heterogeneous.

Group B streptococcal carriage in Sweden: a national study on risk factors for mother and infant colonisation

Background. To study group B streptococcus (GBS) colonisation in parturients and infants in relation to obstetric outcome and to define serotypes and antibiotic resistance in GBS isolates acquired. Methods. A population‐based, national cohort of parturients and their infants was investigated. During 1 calendar week in 2005 all women giving birth (n = 1,754) were requested to participate in the study. Results. A total of 1,569 mother/infant pairs with obstetric and bacteriological data were obtained. Maternal carriage rate was 25.4% (95% confidence interval (CI): 23.3–27.6). In GBS‐positive mothers with vaginal delivery and no intrapartum antibiotics, the infant colonisation rate was 68%. Some 30% of infants were colonised after acute caesarean section, and 0% were colonised after an elective procedure. Duration of transport of maternal recto/vaginal swabs of more than 1 day impeded culture sensitivity. Infant mMales were more frequently colonised than females (76.9 versus 59.8%, odds ratio (OR): 2.16; 95% CI: 1.27–3.70), as were infants born after rupture of membranes ≥24 h (p =0.039). Gestational age, birth weight and duration of labor did not significantly influence infant colonisation. Some 30% of parturients with at least one risk factor for neonatal disease received intrapartum antibiotics. The most common GBS serotypes were type III and V. Some 5% of the isolates were resistant to clindamycin and erythromycin, respectively. Conclusions. Maternal GBS prevalence and infant transfer rate were high in Sweden. Males were more frequently colonised than females. The sensitivity of maternal cultures decreased with the duration of sample transport. Clindamycin resistance was scarce. The use of intrapartum antibiotics was limited in parturients with obstetric risk factors for early onset group B streptococcal disease.

Risk factors for colonization with extended-spectrum beta-lactamase producing Enterobacteriaceae in healthcare students on clinical assignment abroad : A prospective study

BACKGROUND The increase of antibiotic resistance in clinically important bacteria is a worldwide threat, especially in healthcare environments. International travel is a risk factor for gut colonization with extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-PE). The risk for healthcare students of being colonized with ESBL-PE when participating in patient-related work abroad has not been previously investigated. METHODS Swedish healthcare students travelling for pre-clinical and clinical courses outside Scandinavia submitted faecal samples and survey data before and after travel. The faecal samples were screened for ESBL-PE and carbapenemase-producing Enterobacteriaceae (CPE). Screening results and survey data were analysed to identify risk factors for colonization. RESULTS In the 99 subjects who submitted a full set of samples, 35% were colonized with a new ESBL-PE strain during travel. No CPE was found. The most important risk factor for ESBL-PE colonization was travel destination, and the highest colonization rate was found in the South-East Asia region. Antibiotic treatment during travel was an independent risk factor for ESBL-PE colonization but patient-related work was not significantly associated with an increased risk. CONCLUSIONS Patient-related work abroad was not a risk factor for ESBL-PE suggesting that transmission from patients is uncommon. Pre-travel advice on avoiding unnecessary antibiotic treatment during travel is recommended.

Antifungal Application of Nonantifungal Drugs

ABSTRACT Candida species are the cause of 60% of all mycoses in immunosuppressed individuals, leading to ∼150,000 deaths annually due to systemic infections, whereas the current antifungal therapies either have toxic side effects or are insufficiently efficient. We performed a screening of two compound libraries, the Enzo and the Institute for Molecular Medicine Finland (FIMM) oncology collection library, for anti-Candida activity based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. From a total of 844 drugs, 26 agents showed activity against Candida albicans. Of those, 12 were standard antifungal drugs (SADs) and 7 were off-target drugs previously reported to be active against Candida spp. The remaining 7 off-target drugs, amonafide, tosedostat, megestrol acetate, melengestrol acetate, stanozolol, trifluperidol, and haloperidol, were identified with this screen. The anti-Candida activities of the new agents were investigated by three individual assays using optical density, ATP levels, and microscopy. The antifungal activities of these drugs were comparable to those of the SADs found in the screen. The aminopeptidase inhibitor tosedostat, which is currently in a clinical trial phase for anticancer therapy, displayed a broad antifungal activity against different Candida spp., including Candida glabrata. Thus, this screen reveals agents that were previously unknown to be anti-Candida agents, which allows for the design of novel therapies against invasive candidiasis.

CD4 lymphopenia in a patient with cryptococcal osteomyelitis.

Cryptococcus neoformans is a rarely reported cause of osteomyelitis. In most cases, no obvious underlying condition is found. Immunological laboratory data, however, are not generally available. In the present case of cryptococcal osteomyelitis, idiopathic CD4 lymphopenia was detected. This immunodeficiency is found in cases of disseminated cryptococcosis by chance. Possibly, it may be one so far unrecognized underlying condition in cryptococcal osteomyelitis.

Detection of Cryptosporidium and Giardia in clinical laboratories in Europe—a comparative study

To determine the routine diagnostic methods used and compare the performance in detection of oocysts of Cryptosporidium species and cysts of Giardia intestinalis in faecal samples by European specialist parasitology laboratories and European clinical laboratories. Two sets of seven formalin-preserved faecal samples, one containing cysts of Giardia intestinalis and the other, containing oocysts of Cryptosporidium, were sent to 18 laboratories. Participants were asked to examine the specimens using their routine protocol for detecting these parasites and state the method(s) used. Eighteen laboratories answered the questionnaire. For detection of Giardia, 16 of them used sedimentation/concentration followed by light microscopy. Using this technique the lower limit of detection of Giardia was 17.2 cysts/mL of faeces in the best performing laboratories. Only three of 16 laboratories used fluorescent-conjugated antibody-based microscopy. For detection of Cryptosporidium acid-fast staining was used by 14 of the 17 laboratories that examined the samples. With this technique the lower limit of detection was 976 oocysts/mL of faeces. Fluorescent-conjugated antibody-based microscopy was used by only five of the 17 laboratories. There was variation in the lower limit of detection of cysts of Giardia and oocysts of Cryptosporidium between laboratories using the same basic microscopic methods. Fluorescent-conjugated antibody-based microscopy was not superior to light microscopy under the conditions of this study. There is a need for a larger-scale multi-site comparison of the methods used for the diagnosis of these parasites and the development of a Europe-wide laboratory protocol based upon its findings.

An inserted DNA fragment with plasmid features is uniquely associated with the presence of the GBSi1 group II intron in Streptococcus agalactiae

The group II intron (GBSi1) identified downstream of the C5a-peptidase gene (scpB) in a subpopulation of Streptococcus agalactiae isolates is a suggested marker for a separate genetic lineage of serotype III isolates. In the present study two additional copies of GBSi1, one of which not previously described, were identified among serotype III isolates. All intron copies shared a common target site motif. A single copy of GBSi1 was found in a subgroup of serotype II and V isolates. In these isolates, the intron had inserted downstream of scpB, which suggests that this is the primary insertion site for GBSi1. Most bacterial group II introns described to date reside in transposable elements. The scpB locus was found to be flanked by insertion sequences similar to what has been described in an intronless serotype Ia isolate. However, this region contained an additional 2.1 kb DNA fragment present only in intron carrying isolates. This DNA fragment contained a partial transposase and putative plasmid related proteins. This may suggest that GBSi1 once was brought into the S. agalactiae genome by an integrated plasmid.