Discrepancies in Antimicrobial Susceptibility between the JP2 and the Non-JP2 Genotype of Aggregatibacter actinomycetemcomitans.

The Aggregatibacter actinomycetemcomitans JP2 genotype is associated with high leukotoxin production and severe (aggressive) periodontitis. The aim of this study was to compare the antimicrobial susceptibility of JP2 and non-JP2 genotype strains. Minimal inhibitory concentrations (MICs) of 11 antimicrobials were determined for 160 A. actinomycetemcomitans of serotype a, b, or c, mostly isolated in Sweden or Ghana. MIC distributions for benzylpenicillin and fusidic acid revealed a more susceptible subpopulation for 38 serotype b strains, including the 32 of the JP2 genotype, with a benzylpenicillin MIC range of 0.125−0.5 mg/L. In contrast, benzylpenicillin MIC ≤ 16 mg/L was the estimated 99.5% epidemiological cutoff (ECOFF) of all strains. Beta-lactamase production was not detected. The fusidic acid MIC distribution of 11 strains of Aggregatibacter aphrophilus agreed with that found in non-JP2 strains. Cefotaxime, meropenem, levofloxacin, and trimethoprim−sulfamethoxazole MICs were all ≤0.25 mg/L, while MIC90 values for amoxicillin, azithromycin and tetracycline were 1 mg/L. Metronidazole MICs varied between 0.5 and >256 mg/L. The discrepant findings indicate that A. actinomycetemcomitans may be divided into two separate wild types, with a suggested intrinsic reduced susceptibility for benzylpenicillin in the majority of non-JP2 genotype strains. Possible implications for the treatment of A. actinomycetemcomitans infections are discussed.

Harbouring group B streptococci in a neonatal intensive care unit led to an outbreak among preterm infants.

We report a nosocomial outbreak with group B streptococci (GBS) in a level two neonatal intensive care unit (NICU) at Sachs' Children and Youth Hospital, Stockholm, Sweden, in 2014. There were five very preterm infants with severe late-onset septicaemia, and 10 further infants were colonised. Pulsed-field gel electrophoresis and multilocus sequence typing genetic characterisation showed that one GBS strain was the cause: serotype Ia, sequence type 23, clonal complex 23. The NICU environment cultures revealed GBS reservoirs on surfaces near sick and colonised patients. We identified workflows and guidelines that could increase the risks of nosocomial infections. Conclusion: This nosocomial GBS outbreak among preterm infants demonstrates that GBS can be harboured in the NICU environment.

Epidemiology of fungaemia in Sweden: A nationwide retrospective observational survey.

OBJECTIVES: To identify the epidemiology and antifungal susceptibilities of Candida spp. among blood culture isolates to identify the epidemiology and antifungal susceptibilities of Candida spp. among blood culture isolates in Sweden. METHODS: The study was a retrospective, observational nationwide laboratory-based surveillance for fungaemia and fungal meningitis and was conducted from September 2015 to August 2016. RESULTS: In total, 488 Candida blood culture isolates were obtained from 471 patients (58% males). Compared to our previous study, the incidence of candidaemia has increased from 4.2/100 000 (2005-2006) to 4.7/100 000 population/year (2015-2016). The three most common Candida spp. isolated from blood cultures were Candida albicans (54.7%), Candida glabrata (19.7%) and species in the Candida parapsilosis complex (9.4%). Candida resistance to fluconazole was 2% in C. albicans and between 0% and 100%, in non-albicans species other than C. glabrata and C. krusei. Resistance to voriconazole was rare, except for C. glabrata, C. krusei and C. tropicalis. Resistance to anidulafungin was 3.8% while no Candida isolate was resistant to amphotericin B. CONCLUSIONS: We report an overall increase in candidaemia but a minor decrease of C. albicans while C. glabrata and C. parapsilosis remain constant over this 10-year period.

The relation between Blastocystis and the intestinal microbiota in Swedish travellers.

BACKGROUND: Blastocystis sp. is a unicellular eukaryote that is commonly found in the human intestine. Its ability to cause disease is debated and a subject for ongoing research. In this study, faecal samples from 35 Swedish university students were examined through shotgun metagenomics before and after travel to the Indian peninsula or Central Africa. We aimed at assessing the impact of travel on Blastocystis carriage and seek associations between Blastocystis and the bacterial microbiota. RESULTS: We found a prevalence of Blastocystis of 16/35 (46%) before travel and 15/35 (43%) after travel. The two most commonly Blastocystis subtypes (STs) found were ST3 and ST4, accounting for 20 of the 31 samples positive for Blastocystis. No mixed subtype carriage was detected. All ten individuals with a typable ST before and after travel maintained their initial ST. The composition of the gut bacterial community was not significantly different between Blastocystis-carriers and non-carriers. Interestingly, the presence of Blastocystis was accompanied with higher abundances of the bacterial genera Sporolactobacillus and Candidatus Carsonella. Blastocystis carriage was positively associated with high bacterial genus richness, and negatively correlated to the Bacteroides-driven enterotype. These associations were both largely dependent on ST4 - a subtype commonly described from Europe - while the globally prevalent ST3 did not show such significant relationships. CONCLUSIONS: The high rate of Blastocystis subtype persistence found during travel indicates that long-term carriage of Blastocystis is common. The associations between Blastocystis and the bacterial microbiota found in this study could imply a link between Blastocystis and a healthy microbiota as well as with diets high in vegetables. Whether the associations between Blastocystis and the microbiota are resulting from the presence of Blastocystis, or are a prerequisite for colonization with Blastocystis, are interesting questions for further studies.

Actinomyces radicidentis and Actinomyces haliotis, coccoid Actinomyces species isolated from the human oral cavity.

There are few reports on the bacterial species Actinomyces radicidentis in the literature. In this study, putative A. radicidentis isolates were collected from 16 root canal samples from 601 examined patients. The isolates were examined by biochemical tests, 16S rRNA gene sequencing, Arbitrarily-primed (AP-) PCR, antibiotic susceptibility testing, and MALDI-TOF analyses. In parallel, two A. radicidentis reference strains and two putative A. radicidentis isolates from United Kingdom were tested. Sixteen of the 18 isolates were confirmed as A. radicidentis. The remaining two isolates, both of which were isolated from root canals (one from Sweden and the other from the UK), but were identified as Actinomyces haliotis by sequencing âˆ¼ 1300 base pairs of the 16S rRNA-gene. This isolates had a divergent, but between them similar, AP-PCR pattern, and a common distribution of sequence signatures in the 16S rRNA gene, but were not identified by MALDI-TOF. A. haliotis is a close relative to A. radicidentis, hitherto only been described from a sea-snail. The identity of A. haliotis was confirmed by a phylogenetic tree based on 16S rRNA gene sequences with species specific sequences included, and by additional biochemical tests. The examined bacteria exhibited similar antibiotic susceptibility patterns when tested for 10 separate antibiotic classes with E-tests (bioMérieux). The MIC90 for ß-lactams (benzylpenicillin and cefuroxime) and vancomycin was 0.5 mg/L, for colistin and ciprofloxacin 8 mg/mL and for the other antibiotic classes ≤ 25 mg/mL The isolation of A. haliotis from infected dental root canals cast doubt on the accepted opinion that all Actinomyces infections have an endogenous source.

High occurrence of Blastocystis sp. subtypes 1-3 and Giardia intestinalis assemblage B among patients in Zanzibar, Tanzania.

BACKGROUND: Blastocystis is a common intestinal parasite with worldwide distribution but the distribution of Blastocystis and its subtypes in East Africa is largely unknown. In this study, we investigate the distribution of Blastocystis subtypes in Zanzibar, Tanzania and report the prevalence of intestinal parasites using both molecular methods and microscopy. METHODS: Stool samples were collected from both diarrhoeic and non-diarrhoeic outpatients in Zanzibar. In addition to microscopy, real-time PCR for Blastocystis, Entamoeba histolytica and E. dispar, Giardia intestinalis, Cryptosporidium spp., and Dientamoeba fragilis was used. Blastocystis subtypes were determined by a conventional PCR followed by partial sequencing of the SSU-rRNA gene. Genetic assemblages of Giardia were determined by PCR with assemblage specific primers. RESULTS: Intestinal parasites were detected in 85 % of the 174 participants, with two or more parasites present in 56 %. Blastocystis sp. and Giardia intestinalis were the most common parasites, identified by PCR in 61 and 53 % of the stool samples respectively, but no correlation between carriage of Blastocystis and Giardia was found. The Blastocystis subtype distribution was ST1 34.0 %, ST2 26.4 %, ST3 25.5 %, ST7 0.9 %, and 13.2 % were positive only by qPCR (non-typable). The Giardia genetic assemblages identified were A 6.5 %, B 85 %, A + B 4.3 %, and non-typable 4.3 %. The detection rate with microscopy was substantially lower than with PCR, 20 % for Blastocystis and 13.8 % for Giardia. The prevalence of Blastocystis increased significantly with age while Giardia was most prevalent in children two to five years old. No correlation between diarrhoea and the identification of Giardia, Blastocystis, or their respective genetic subtypes could be shown and, as a possible indication of parasite load, the mean cycle threshold values in the qPCR for Giardia were equal in diarrhoeic and non-diarrhoeic patients. CONCLUSIONS: Carriage of intestinal parasites was very common in the studied population in Zanzibar. The most commonly detected parasites, Blastocystis and Giardia, had different age distributions, possibly indicating differences in transmission routes, immunity, and/or other host factors for these two species. In the Blastocystis subtype analysis ST1-3 were common, but ST4, a subtype quite common in Europe, was completely absent, corroborating the geographical differences in subtype distributions previously reported.

Evaluation of factors affecting real-time PCR performance for diagnosis of Entamoeba histolytica and Entamoeba dispar in clinical stool samples.

Although PCR offers the potential for sensitive detection of parasites there are several pitfalls for optimal performance, especially when DNA is extracted from a complex sample material such as stool. With the aid of a sensitive inhibitor control in a duplex real-time PCR (qPCR) for identification of Entamoeba histolytica and Entamoeba dispar we have evaluated factors that influenced the performance of the qPCR and have suggested a rationale to be used in the analysis of clinical samples. Pre-PCR processing was found to be of outmost importance for an optimal amplification since inhibitors caused false-negative results when higher amounts of sample were used. Stool sampling with a flocked swab (ESwab, Copan), yielding on average 173 mg, gave positive qPCR results in samples with cysts of E. dispar that were negative in serially diluted stool samples. The degree of inhibition found varied between samples and was not an on-off phenomenon. Even low-grade inhibition, shown as an increase of two cycles in the qPCR for the inhibitor control, could lead to false negativity in samples with low amounts of parasites. Lack of amplification in the qPCR due to inhibition could be overcome by dilution of the extracted DNA by 1/10-1/20. We also describe the use of guanidinium thiocyanate buffer for transport and storage of samples as well as a time-saving semi-automated DNA extraction method in an Arrow instrument (Nordiag) preceded by bead beating.

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.

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.

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 > or =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.

Multilocus sequence typing of Swedish invasive group B streptococcus isolates indicates a neonatally associated genetic lineage and capsule switching.

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.

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.

Stomatococcus mucilaginosus septicemia in leukemic patients.

OBJECTIVE: To report an unexpectedly high number of cases of septicemia with Stomatococcus mucilaginosus, and try to identify predisposing factors. METHODS: All blood cultures obtained during 1991--93 from patients treated at the hematologic ward were bacteriologically identified. The medical records of patients with S. mucilaginosus-positive blood cultures were retrospectively reviewed and evaluated. The antibiotic susceptibility pattern and restriction fragment length polymorphism (RFLP) of S. mucilaginosus were tested. RESULTS: S. mucilaginosus blood isolates from patients with hematologic malignancies were found to be as common as isolates of Staphylococcus aureus. Eleven patients with myelogenous leukemia and isolation of S. mucilaginosus from the blood are reported on. One patient had concomitant meningitis. All patients were neutropenic and most had oral mucositis and had been given ciprofloxacin prophylaxis. S. mucilaginosus isolates from these patients were resistant to ciprofloxacin in contrast to isolates from patients who had received other prophylactic regimens and seven isolates found in healthy individuals not recently treated with antibiotics. The resistant S. mucilaginosus were found to be of diverse genetic origin as determined by RFLP. CONCLUSIONS: The appearance of resistant strains during ciprofloxacin prophylaxis may be a predisposing factor for S. mucilaginosus septicemia. There was no evidence of a nosocomial spread of S. mucilaginosus strains.