Characterization and Fitness Cost of Tn7100, a Novel Integrative and Conjugative Element Conferring Multidrug Resistance in Haemophilus influenzae.

A multidrug-resistant (MDR) strain of Haemophilus influenzae, Hi-228, with phenotypic resistance toward ampicillin, cefotaxime, chloramphenicol, gentamicin, and azithromycin, was isolated in Oslo, Norway. The strain was part of a clonal outbreak (2016-2017) comprising five ST143 strains with identical resistotypes. Hi-228 carries a novel integrative and conjugative element (ICE), Tn7100, contributing to this remarkable and previously unreported MDR profile. Tn7100 contains the following resistance genes: bla TEM-1B, catA2, aac(6')-Im, aph(2″)-Ib, mef (E), and mel. The latter four are previously unreported or rarely reported in H. influenzae. In this study, we investigated the genetic environment, mechanisms of transfer, impact on phenotypic susceptibility, and fitness cost of this ICE. We found that Tn7100 has an overall structure similar to the previously described ICE Tn6686, with bla TEM-1B and catA2 carried by Tn3 and Tn10, respectively. The major difference between Tn7100 and Tn6686 is that Tn7100 lacks tet(B) but carries the resistance gene pairs aac(6')-Im and aph(2″)-Ib and mef (E) and mel. The gene pairs are located on the novel transposable elements Tn7470 and Tn7471, which have high sequence identities to a plasmid in Enterobacterales and an ICE in streptococcal species, respectively. Tn7100 does circularize and is transferable, however, at a low frequency. Head-to-head competition experiments showed that uptake of Tn7100 reduces bacterial fitness. Our study shows that MDR strains are capable of clonal spread and that the H. influenzae supragenome comprises an increasingly wide range of transferable resistance genes, with evidence of transfer from unrelated genera. The findings offer a glimpse into the genome dynamics of H. influenzae, highlighting the importance of rational antibiotic usage to contain antimicrobial resistance and the emergence of MDR strains in this important pathogen.

Rapid diagnosis and reduced workload for urinary tract infection using flowcytometry combined with direct antibiotic susceptibility testing.

BACKGROUND: We evaluated if flowcytometry, using Sysmex UF-5000, could improve diagnosis of urinary tract infections by rapid identification of culture negative and contaminated samples prior to culture plating, thus reducing culture plating workload and response time. We also evaluated if it is possible to reduce the response time for antibiotic susceptibility profiles using the bacteria information flag on Sysmex UF-5000 to differentiate between Gram positive and negative bacteria, followed by direct Antibiotic Susceptibility Testing (dAST) on the positive urine samples. METHODS: One thousand urine samples were analyzed for bacteria, white blood cells and squamous cells by flowcytometry before culture plating. Results from flowcytometric analysis at different cut-off values were compared to results of culture plating. We evaluated dAST on 100 urine samples that were analyzed as positive by flowcytometry, containing either Gram positive or Gram negative bacteria. RESULTS: Using a cut-off value with bacterial count ≥100.000/mL and WBCs ≥10/µL, flowcytometry predicted 42,1% of samples with non-significant growth. We found that most contaminated samples contain few squamous cells. For 52/56 positive samples containing Gram negative bacteria dAST was identical to routine testing. Overall, there was concordance in 555/560 tested antibiotic combinations. CONCLUSION: Flowcytometry offers advantages for diagnosis of urinary tract infections. Screening for negative urine samples on the day of arrival reduces culture plating and workload, and results in shorter response time for the negative samples. The bacteria information flag predicts positive samples containing Gram negative bacteria for dAST with high accuracy, thus Antibiotic Susceptibility Profile can be reported the day after arrival. For the positive samples containing Gram negative bacteria the concordance was very good between dAST and Antibiotic Susceptibility Testing in routine. For positive samples containing Gram positive bacteria the results were not convincing. We did not find any correlation between epithelial cells and contamination.

Molecular epidemiology of metallo-beta-lactamase-producing Pseudomonas aeruginosa isolates from Norway and Sweden shows import of international clones and local clonal expansion.

Scandinavia is considered a region with a low prevalence of antimicrobial resistance. However, the number of multidrug-resistant (MDR) Gram-negative bacteria is increasing, including metallo-beta-lactamase (MBL)-producing Pseudomonas aeruginosa. In this study MBL-producing P. aeruginosa isolates identified in Norway (n = 4) and Sweden (n = 9) from 1999 to 2007 were characterized. Two international clonal complexes (CC), CC111 (n = 8) and CC235 (n = 2), previously associated with MBL-producing isolates, were dominant. CC111 isolates (ST111/229; serotype O12; bla(VIM-2)) included clonally related isolates identified in Skåne County, Sweden (n = 6), and two isolates associated with importation from Greece and Denmark. In all CC111 isolates, bla(VIM-2) was located in integron In59.2 or In59 variants. The two CC235 isolates (ST235/ST230; serotype O11; bla(VIM-4)) were imported from Greece and Cyprus, were possibly clonally related, and carried bla(VIM-4) in two different integron structures. Three isolates imported from Ghana (ST233; serotype O6; bla(VIM-2)), Tunisia (ST654; serotype O11; bla(VIM-2)), and Thailand (ST260; serotype O6; bla(IMP-14)) were clonally unrelated. ST233 was part of a new CC (CC233) that included other MBL-producing isolates, while ST654 could also be part of a new CC associated with MBL producers. In the isolates imported from Ghana and Tunisia, bla(VIM-2) was part of unusual integron structures lacking the 3' conserved segment and associated with transposons. The bla(VIM) gene was found to be located on the chromosome in all isolates. Known risk factors for acquisition of MBL were reported for all patients except one. The findings suggest that both import of successful international clones and local clonal expansion contribute to the emergence of MBL-producing P. aeruginosa in Scandinavia.

The impact of age on risk assessment, therapeutic practice and outcome in candidemia.

BACKGROUND: In Norway, the epidemiological situation of candidemia is followed closely. We have previously demonstrated the highest incidence of candidemia in elderly >65 years of age. However, knowledge of other aspects of this infection is lacking. OBJECTIVE: The aim of this nationwide, retrospective study was to examine risk factors, therapeutic practice and outcome in adult candidemia patients according to age. METHODS: We retrieved data from medical records from patients who developed candidemia in Norway between 1 January 2008 and 31 December 2012. Data were analyzed according to age, younger patients being between 18 and 65 years, elderly being ≥65 years of age. RESULTS: From 771 eligible patients, 738 patients (95.7%) were included (58% men, mean age 65.2 years, 58.1% being ≥65 years). Exposure to health-care related risk factors for candidemia were significantly more common in the younger patients (neutropenia, central venous catheter, mechanical ventilation and chemotherapy) who received empirical treatment more often than the elderly (29.8% vs. 21.7%, p = .01). More elderly did not received any antifungal therapy (27.3% vs 16.8%, p < 0001) and had higher mortality compared to younger patients (45.5% vs 23.9%, p < .0001). In the study population, mortality was higher with age (per 10-years increase, OR 1.43;1.28-1.59, p < 0.0001), in patients not receiving targeted therapy (OR 2.5; CI 1.82-3.36, p < .0001) or any therapy at all (OR 4.64; 3.23-6.68, p < .0001). CONCLUSIONS: Risk factors for candidemia, treatment and outcome differed significantly according to age. Given the increasing numbers of elderly, scrutiny on our clinical practice is warranted.

Suitability of stx-PCR directly from fecal samples in clinical diagnostics of STEC.

PCR-based testing for Shiga toxin producing Escherichia coli (STEC) directly from fecal samples is increasingly being implemented in routine diagnostic laboratories. These methods aim to detect clinically relevant amounts of microbes and not stx-carrying phages or low backgrounds of STEC. We present a diagnostic procedure and results from 1 year of stx-targeted real-time PCR of fecal samples from patients with gastrointestinal symptoms in Norway. A rapid stx2 subtyping strategy is described, which aims to quickly reveal the virulence potential of the microbe. stx was detected in 22 of 3320 samples, corresponding to a PCR positive rate of 0.66%. STEC were cultured from 72% of the PCR positive samples. Four stx1 isolates, eight stx2 isolates, and four isolates with both stx1 and stx2 were identified. With the method presented, stx-carrying phages are not commonly detected. Our results support the use of molecular testing combined with classical culture techniques for routine diagnostic purposes.

Nosocomial candidemia; risk factors and prognosis revisited; 11 years experience from a Norwegian secondary hospital.

The aim of the study was to review the epidemiology and prognosis of candidemia in a secondary hospital, and to examine the intra-hospital distribution of candidemia patients. Study design is a retrospective cohort study. Trough 2002-2012, 110 cases of candidemia were diagnosed, giving an incidence of 2, 6/100,000 citizens/year. Overall prognosis of candidemia was dismal, with a 30 days case fatality rate of 49% and one year case fatality rate of 64%. Candidemia was a terminal event in 55% of 30 days non-survivors, defined as Candida blood cultures reported positive on the day of death or thereafter (39%), or treatment refrained due to hopeless short-term prognosis (16%). In terminal event candidemias, advanced or incurable cancer was present in 29%. Non-survivors at 30 days were 9 years (median) older than survivors. In 30 days survivors, candidemia was not recognised before discharge in 13% of cases. No treatment were given and no deaths or complications were observed in this group. Candidemia patients were grouped into 8 patient categories: Abdominal surgery (35%), urology (13%), other surgery (11%), pneumonia (13%), haematological malignancy (7%), intravenous drug abuse (4%), other medical (15%), and new-borns (3%). Candidemia was diagnosed while admitted in the ICU in 46% of patients. Urology related cases were all diagnosed in the general ward. Multiple surgical procedures were done in 60% of abdominal surgery patients. Antibiotics were administered prior to candidemia in 87% of patients, with median duration 17 (1-108) days. Neutropenia was less common than expected in patients with candidemia (8/105) and closely associated to haematological malignancy (6/8). Compared with previous national figures the epidemiology of invasive candidiasis seems not to have changed over the last decade.

Exflagellation of microgametocytes in Plasmodium vivax malaria: a diagnostic conundrum.

OBJECTIVE: To present a clinical diagnostic conundrum of unidentified structures in a blood smear from a patient with Plasmodium vivax malaria. CLINICAL PRESENTATION AND INTERVENTION: A 37-year-old Ethiopian male presented with a 4-month history of chills, chronic diarrhea and weight loss. He was diagnosed with P. vivax malaria, advanced HIV infection and Isospora belli enteritis. Unidentified structures initially confusing to the diagnosticians were seen in blood smears taken on admission. The structures were initially considered to represent atypical spirochetes, but were later identified as microgametes and other exflagellation forms of P. vivax. The patient recovered after receiving adequate treatment for his infections. CONCLUSION: This case illustrates that exflagellation may be observed in blood smears from patients with P. vivax malaria. Size and morphological characteristics differentiate malaria microgametes and other exflagellation forms from microfilaria, spirochetes and trypanosomes.