Background. Broad-range 16S ribosomal RNA (rRNA) gene polymerase chain reaction (PCR) is used for detection and identification of bacterial pathogens in clinical specimens from patients with a high suspicion for infection. However, prospective studies addressing the impact and clinical value of broad-range bacterial 16S rRNA gene amplification for diagnosis of acute infectious diseases in nonselected patient populations are lacking. Methods. We first assessed the diagnostic performance of 16S rRNA gene PCR compared with routine bacterial culture. Second, we addressed prospectively the impact and clinical value of broad-range PCR for the diagnosis of acute infections using samples that tested negative by routine bacterial culture; the corresponding patients’ data were evaluated by detailed medical record reviews. Results. Results from 394 specimens showed a high concordance of >90% for 16S rRNA gene PCR and routine bacterial culture, indicating that the diagnostic performance of PCR for acute bacterial infections is comparable to that of bacterial culture, which is currently considered the gold standard. In thisprospective study, 231 specimens with a negative result on routine bacterial culture were analyzed with PCR, and patients’ clinical data were reviewed. We found that broad-range 16S rRNA gene PCR showed a sensitivity, specificity, positive predictive value, and negative predictive value of 42.9%, 100%, 100%, and 80.2% for culture-negative bacterial infections. Conclusions. This study defines the role of 16S rRNA gene PCR for diagnosis of culture-negative bacterial infections. Our data show that 16S rRNA gene PCR is particularly useful for identification of bacterial pathogens in patients pretreated with antibiotics.

The risk of tuberculosis outbreaks among people fleeing hardship for refuge in Europe is heightened. We describe the cross-border European response to an outbreak of multidrug-resistant tuberculosis among patients from the Horn of Africa and Sudan. On April 29 and May 30, 2016, the Swiss and German National Mycobacterial Reference Laboratories independently triggered an outbreak investigation after four patients were diagnosed with multidrug-resistant tuberculosis. In this molecular epidemiological study, we prospectively defined outbreak cases with 24-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) profiles; phenotypic resistance to isoniazid, rifampicin, ethambutol, pyrazinamide, and capreomycin; and corresponding drug resistance mutations. We whole-genome sequenced all Mycobacterium tuberculosis isolates and clustered them using a threshold of five single nucleotide polymorphisms (SNPs). We collated epidemiological data from host countries from the European Centre for Disease Prevention and Control. Between Feb 12, 2016, and April 19, 2017, 29 patients were diagnosed with multidrug-resistant tuberculosis in seven European countries. All originated from the Horn of Africa or Sudan, with all isolates two SNPs or fewer apart. 22 (76%) patients reported their travel routes, with clear spatiotemporal overlap between routes. We identified a further 29 MIRU-VNTR-linked cases from the Horn of Africa that predated the outbreak, but all were more than five SNPs from the outbreak. However all 58 isolates shared a capreomycin resistance-associated tlyA mutation. Our data suggest that source cases are linked to an M tuberculosis clone circulating in northern Somalia or Djibouti and that transmission probably occurred en route before arrival in Europe. We hypothesise that the shared mutation of tlyA is a drug resistance mutation and phylogenetic marker, the first of its kind in M tuberculosis sensu stricto. The Swiss Federal Office of Public Health, the University of Zurich, the Wellcome Trust, National Institute for Health Research (NIHR) Oxford Biomedical Research Centre (BRC), the Medical Research Council, BELTA-TBnet, the European Union, the German Center for Infection Research, and Leibniz Science Campus Evolutionary Medicine of the Lung (EvoLUNG).

Helicobacter pylori is a major human pathogen. Diagnosis of H. pylori infection and determination of its antibiotic susceptibility still mainly rely on culture and phenotypic drug susceptibility testing (DST) that is time-consuming and laborious. Whole genome sequencing (WGS) has recently emerged in medical microbiology as a diagnostic tool for reliable drug resistance prediction in bacterial pathogens. The aim of this study was to compare phenotypic DST results with the predictions based on the presence of genetic determinants identified in the H. pylori genome using WGS. Phenotypic resistance to clarithromycin, metronidazole, tetracycline, levofloxacin, and rifampicin was determined in 140 clinical H. pylori isolates by E-Test^®, and the occurrence of certain single nucleotide polymorphisms (SNPs) in target genes was determined by WGS. Overall, there was a high congruence of >99% between phenotypic DST results for clarithromycin, levofloxacin, and rifampicin and SNPs identified in the 23S rRNA, gyrA, and rpoB gene. However, it was not possible to infer a resistance phenotype for metronidazole based on the occurrence of distinct SNPs in frxA and rdxA. All 140 H. pylori isolates analysed in this study were susceptible to tetracycline, which was in accordance with the absence of double or triple nucleotide substitutions in the 16S rRNA gene.

We have repeatedly detected Candidatus Neoehrlichia mikurensis, a bacterium first described in Rattus norvegicus rats and Ixodes ovatus ticks in Japan in 2004 in the blood of a 61-year-old man with signs of septicemia by 16S rRNA and groEL gene PCR. After 6 weeks of therapy with doxycycline and rifampin, the patient recovered.

Since cations have been reported as essential regulators of biofilm, we investigated the potential of the broad-spectrum antimicrobial and cation-chelator nitroxoline as an antibiofilm agent. Biofilm mass synthesis was reduced by up to 80% at sub-MIC nitroxoline concentrations in Pseudomonas aeruginosa , and structures formed were reticulate rather than compact. In preformed biofilms, viable cell counts were reduced by 4 logs at therapeutic concentrations. Complexation of iron and zinc was demonstrated to underlie nitroxoline's potent antibiofilm activity.

Rationale: In cystic fibrosis (CF), the paranasal sinuses are sites of first and persistent colonization by pathogens such as Pseudomonas aeruginosa. Pathogens subsequently descend to the lower airways, with P. aeruginosa remaining the primary cause of premature death in patients with the inherited disease. Unlike conventional aerosols, vibrating aerosols applied with the PARI Sinus™ nebulizer deposit drugs into the paranasal sinuses. This trial assessed the effects of vibrating sinonasal inhalation of the antibiotic tobramycin in CF patients positive for P. aeruginosa in nasal lavage. Objectives: To evaluate the effects of sinonasal inhalation of tobramycin on P. aeruginosa quantification in nasal lavage; and on patient quality of life, measured with the Sino-Nasal Outcome Test (SNOT-20), and otologic and renal safety and tolerability. Methods: Patients were randomized to inhalation of tobramycin (80 mg/2 mL) or placebo (2 mL isotonic saline) once daily (4 minutes/nostril) with the PARI Sinus™ nebulizer over 28 days, with all patients eligible for a subsequent course of open-label inhalation of tobramycin for 28 days. Nasal lavage was obtained before starting and 2 days after the end of each treatment period by rinsing each nostril with 10 mL of isotonic saline. Results: Nine patients participated, six initially receiving tobramycin and three placebo. Sinonasal inhalation was well tolerated, with serum tobramycin <0.5 mg/L and stable creatinine. P. aeruginosa quantity decreased in four of six (67%) patients given tobramycin, compared with zero of three given placebo (non-significant). SNOT-20 scores were significantly lower in the tobramycin than in the placebo group (P=0.033). Conclusion: Sinonasal inhalation of vibrating antibiotic aerosols appears promising for reducing pathogen colonization of paranasal sinuses and for control of symptoms in patients with CF.