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AIMS OF THE STUDY: Numerous studies from different countries have contributed to an improved understanding of blood culture-negative infective endocarditis. However, little is known about its epidemiology and microbiology in Switzerland. We aimed to assess the epidemiology and microbiology of blood culture-negative endocarditis at the University Hospital Zurich, Switzerland. METHODS: We screened all patients hospitalised between 1997 and 2020 with possible or definite endocarditis at our institution. Thereof, we identified all cases with blood culture-negative endocarditis and retrospectively retrieved patient characteristics, microbiological, histopathological, radiographic and surgical data from medical records. RESULTS: Among 861 patients screened, 66 (7.7%) cases of blood culture-negative endocarditis were identified. Thereof, 31 cases could be microbiologically documented or not documented (n = 30), and in five cases a non-infectious aetiology was confirmed. Endocarditis predominantly affected men (77%) and the left heart (79%); predisposing factors were prosthetic valves (42%), congenital heart disease (35%) and prior endocarditis (14%). The most common reasons for negative blood cultures were antibiotic treatment prior to blood culture sampling (35%), fastidious and slow growing microorganisms (30%) and definite non-infective endocarditis (8%). Coxiella burneti i and Bartonella spp. were the most common fastidious bacteria identified. In addition to serology, identification of causative microorganisms was possible by microbiological and/or histopathological analysis of tissue samples, of which polymerase chain reaction testing (PCR) of the 16S ribosomal RNA proved to be most successful. CONCLUSIONS: The present study provides a detailed analysis of blood culture-negative endocarditis over a time span of more than 20 years in Zurich, Switzerland. Antibiotic treatment prior to blood collection, and fastidious and slow growing organisms were identified as main reasons for sterile blood cultures. Typical culture-negative bacteria were mainly found by PCR and/or culture of tissue samples.

Nocardiosis is known to be an opportunistic infection most commonly affecting immunocompromised patients that can lead to life-threatening conditions. Primary cutaneous disease remains a rare manifestation and unlike pulmonary or disseminated nocardiosis, it usually affects immunocompetent individuals. We present a case of a primary cutaneous nocardiosis of the head and neck after an insect bite in a healthy 50-year-old woman who had recently travelled from Greece. She presented with a painful right-sided swelling of her face and neck and an ulcerated plaque over the right temple. Biopsy of the plaque revealed inflammation with abscess formation indicating underlying infection. Culture from the biopsy showed growth ofNocardiaspp and 16S rRNA gene sequence analysis identifiedNocardia brasiliensis. The patient was treated with trimethoprim/sulfamethoxazole and subsequently switched to amoxicillin/clavulanic acid due to a drug eruption. Antibiotic therapy was continued for a total of 3 months with complete resolution of the skin lesions.

SUMMARY Objective The international outbreak of cardiac-surgery-associated Mycobacterium chimaera infections was traced back to infectious aerosols originating from contaminated water reservoirs of heater-cooler devices (HCDs). In general, non-tuberculous mycobacteria (NTM) frequently colonize water systems and can contaminate medical devices. Data on detection of NTM other than M. chimaera in samples gathered from HCDs are scarce. The present study summarizes prospective mycobacterial surveillance of five HCDs over more than 4 years. Methods Five LivaNova 3T (London, UK) HCDs, acquired factory-new in 2014, were followed prospectively. Until mid-April 2014, the HCDs were maintained according to the manufacturer's recommendations, and subsequently, they were maintained according to an intensified in-house protocol including exhaust air evacuation. Mycobacterial surveillance cultures consisted of monthly water samples gathered from patient and cardioplegia circuits, as well as airflow samples. Results Of 441 water samples, 170 (38.6%) revealed NTM growth. The most frequently detected NTM were M. chimaera [N=120 (67.4%)], Mycobacterium gordonae [N=35 (19.7 %)] and Mycobacterium paragordonae [N=17 (9.6%)]. Growth of NTM, M. chimaera and M. paragordonae was significantly more common in water samples derived from the patient circuit than the cardioplegia circuit of the HCD. Three (2.0%) of 150 air samples grew NTM. Conclusion Growth of NTM in HCD water samples was common. Diverse NTM species were detected, with M. chimaera being the most common. The majority of air samples remained negative. The relevance of NTM other than M. chimaera contaminating HCDs is poorly defined, but a recent report on an HCD-associated outbreak with Mycobacterium abscessus confirmed a potential threat.

The co-occurrence of aminoglycoside and β-lactam resistance was assessed in 3358 consecutive Escherichia coli clinical isolates collected in 2014 in the greater Zurich area, Switzerland. Non-susceptibility to at least one of the tested aminoglycosides was observed in 470/3358 E. coli strains (14%). In strains categorized as broad-spectrum β-lactamase (BSBL)-producers (1241/3358 isolates), extended-spectrum β-lactamase (ESBL)-producers (262/3358) and AmpC-producers (66/3358), resistance to aminoglycoside was found in 23%, 52% and 20% of the isolates, respectively. In contrast, aminoglycoside-susceptible strains were rarely resistant to β-lactams (33/1777, 1.9%). The genomes of 439 aminoglycoside-resistant E. coli were sequenced and aminoglycoside and β-lactam genotypes were analysed. The most prevalent aminoglycoside resistance genes were aph(3')-Ia (133 strains, 30.3%), aac(3)-IId (100 strains, 22.8%), and aac(6')-Ib-cr (52 strains, 11.8%). The most frequent associations with β-lactam resistance genes were aph(3')-Ia or aac(3)-IId with blaTEM-1 (94 and 72 strains, respectively), and aac(3)-IIa/aac(6')-Ib-cr with blaCTX-M-15/blaOXA-1 (23 strains). These results indicate a frequent association of aac(3) and aph(3') genotypes with BSBL production, and a frequent co-occurrence of aac(6') genes with ESBL production. The high rate of co-occurrence of aminoglycoside resistance and β-lactamase production must be considered in combination therapy.

A novel New Delhi metallo-β-lactamase (NDM) variant, NDM-19, was identified in a carbapenem-resistant E. coli strain isolated from a subcutaneous infection of a laparotomy scar from an Egyptian patient in a Swiss hospital. NDM-19 is a derivative of NDM-7, from which it differs by a single amino acid substitution (Ala233Val). Under zinc-limiting growth conditions, E. coli DH5α transformants producing NDM-19 displayed reduced susceptibility towards expanded-spectrum cephalosporins and carbapenems as compared to transformants producing NDM-1 or NDM-7.

Helicobacter pylori is a major human pathogen that causes a wide range of gastrointestinal pathology. Progression of H. pylori induced gastritis to more severe disease has been found to highly correlate with the array of virulence factors expressed by the pathogen. The objective of this study was twofold: first, to characterize the genetic diversity of H. pylori strains isolated from 41 non-atrophic gastritis patients in Switzerland, an issue that has not been investigated to date. And second, to assess the prevalence and sequence variation of H. pylori virulence factors (cagA, vacA, iceA and dupA) and genes encoding outer membrane proteins (OMPs, babA, babB, sabA, sabB, hopZ, hopQ and oipA) by whole genome sequencing (WGS) using an Illumina MiSeq platform. WGS identified high genetic diversity in the analyzed H. pylori strains. Most H. pylori isolates were assigned to hpEurope (95.0%, 39/41), and the remaining ones (5.0%, 2/41) to hpEastAsia, subpopulation hspEAsia. Analysis of virulence factors revealed that 43.9% of the strains were cagA-positive, and the vacA s1 allele was detected in 56.0% of the isolates. The presence of cagA was found to be significantly associated (P &lt, 0.001) with the presence of vacA s1, babA2 and hopQ allele 1 as well as expression of oipA. Moreover, we found an association between the grade of gastritis and H. pylori abundance in the gastric mucosa, respectively and the presence of cagA, vacA s1 and hopQ allele 1. Among our 41 gastritis patients, we identified seven patients infected with H. pylori strains that carried a specific combination of virulence factors (i.e cagA, vacA s1 allele and babA2 allele), recently implicated in the development of more severe gastrointestinal pathology, like peptic ulcer disease and even gastric cancer. To this end, WGS can be employed for rapid and detailed characterization of virulence determinants in H. pylori, providing valuable insights into the pathogenic capacity of the bacterium. This could ultimately lead to a higher level of personalized treatment and management of patients suffering from H. pylori associated infections.

Background Interpretative reading of antimicrobial susceptibility test (AST) results allows inferring biochemical resistance mechanisms from resistance phenotypes. For aminoglycosides, however, correlations between resistance pathways inferred on the basis of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints and expert rules versus genotypes are generally poor. This study aimed at developing and validating a decision tree based on resistance phenotypes determined by disc diffusion and based on epidemiological cut-offs (ECOFFs) to infer the corresponding resistance mechanisms in Escherichia coli. Methods Phenotypic antibiotic susceptibility of thirty wild-type and 458 aminoglycoside-resistant E. coli clinical isolates was determined by disc diffusion and the genomes were sequenced. Based on well-defined cut-offs, we developed a phenotype-based algorithm (Aminoglycoside Resistance Mechanism Inference Algorithm - ARMIA) to infer the biochemical mechanisms responsible for the corresponding aminoglycoside resistance phenotypes. The mechanisms inferred from susceptibility to kanamycin, tobramycin and gentamicin were analysed using ARMIA- or EUCAST-based AST interpretation and validated by whole genome sequencing (WGS) of the host bacteria. Findings ARMIA-based inference of resistance mechanisms and WGS data were congruent in 441/458 isolates (96·3%). In contrast, there was a poor correlation between resistance mechanisms inferred using EUCAST CBPs/expert rules and WGS data (418/488, 85·6%). Based on the assumption that resistance mechanisms can result in therapeutic failure, EUCAST produced 63 (12·9%) very major errors (vME), compared to only 2 (0·4%) vME with ARMIA. When used for detection and identification of resistance mechanisms, ARMIA resolved >95% vMEs generated by EUCAST-based AST interpretation. Interpretation This study demonstrates that ECOFF-based analysis of AST data of only four aminoglycosides provides accurate information on the resistance mechanisms in E. coli. Since aminoglycoside resistance mechanisms, despite having in certain cases a minimal effect on the minimal inhibitory concentration, may compromise the bactericidal activity of aminoglycosides, prompt detection of resistance mechanisms is crucial for therapy. Using ARMIA as an interpretative rule set for editing AST results allows for better predictions of in vivo activity of this drug class., eBioMedicine, 46, ISSN:2352-3964

Objectives Rapid detection of macrolide resistance–associated mutations in Mycoplasma pneumoniae is crucial for effective antimicrobial treatment. We evaluated the Lightmix Mycoplasma macrolide assay for the detection of point mutations at nucleotide positions 2063 and 2064 in the 23S ribosomal RNA (rRNA) gene of M. pneumoniae that confer macrolide resistance. Methods Samples from 3438 patients with a respiratory tract infection were analysed by M. pneumoniae real-time PCR, and 208 (6%) of them were tested positive. In this retrospective study, 163 M. pneumoniae real-time PCR–positive samples were analysed by the Lightmix assay, and results were compared to targeted 23S rRNA sequencing. Results Macrolide-resistant M. pneumoniae were found in 15 (9%) of 163 retrospectively analysed samples. The Lightmix assay showed a sensitivity of 100% (95% confidence interval, 78.2–100) and a specificity of 100% (95% confidence interval, 97.5–100) as the detected M. pneumoniae genotype (148 wild type and 15 non–wild type) was confirmed by 23S rRNA sequencing in all samples. Conclusions The Lightmix assay is an easy-to-use and accurate molecular test that allows rapid determination of macrolide resistance in M. pneumoniae.

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&reg, and the occurrence of certain single nucleotide polymorphisms (SNPs) in target genes was determined by WGS. Overall, there was a high congruence of &gt, 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.

Pneumonia is a severe infectious disease. In addition to common viruses and bacterial pathogens (e.g. Streptococcus pneumoniae), fastidious respiratory pathogens like Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella spp. can cause severe atypical pneumonia. They do not respond to penicillin derivatives, which may cause failure of antibiotic empirical therapy. The same applies for infections with B. pertussis and B. parapertussis, the cause of pertussis disease, that may present atypically and need to be treated with macrolides. Moreover, these fastidious bacteria are difficult to identify by culture or serology, and therefore often remain undetected. Thus, rapid and accurate identification of bacterial pathogens causing atypical pneumonia is crucial. We performed a retrospective method evaluation study to evaluate the diagnostic performance of the new, commercially available Lightmix ® multiplex RT-PCR assay that detects these fastidious bacterial pathogens causing atypical pneumonia. In this retrospective study, 368 clinical respiratory specimens, obtained from patients suffering from atypical pneumonia that have been tested negative for the presence of common agents of pneumonia by culture and viral PCR, were investigated. These clinical specimens have been previously characterized by singleplex RT-PCR assays in our diagnostic laboratory and were used to evaluate the diagnostic performance of the respiratory multiplex Lightmix ® RT-PCR. The multiplex RT-PCR displayed a limit of detection between 5 and 10 DNA copies for different in-panel organisms and showed identical performance characteristics with respect to specificity and sensitivity as in-house singleplex RT-PCRs for pathogen detection. The Lightmix ® multiplex RT-PCR assay represents a low-cost, time-saving and accurate diagnostic tool with high throughput potential. The time-to-result using an automated DNA extraction device for respiratory specimens followed by multiplex RT-PCR detection was below 4 h, which is expected to significantly improve diagnostics for atypical pneumonia-associated bacterial pathogens.

Bacteria make use of compartmentalizing protease complexes, similar in architecture but not homologous to the eukaryotic proteasome, for the selective and processive removal of proteins. Mycobacteria as members of the actinobacteria harbor proteasomes in addition to the canonical bacterial degradation complexes. Mycobacterial proteasomal degradation, although not essential during normal growth, becomes critical for survival under particular environmental conditions, like, for example, during persistence of the pathogenic Mycobacterium tuberculosis in host macrophages or of environmental mycobacteria under starvation. Recruitment of protein substrates for proteasomal degradation is usually mediated by pupylation, the post-translational modification of lysine side chains with the prokaryotic ubiquitin-like protein Pup. This substrate recruitment strategy is functionally reminiscent of ubiquitination in eukaryotes, but is the result of convergent evolution, relying on chemically and structurally distinct enzymes. Pupylated substrates are recognized by the ATP-dependent proteasomal regulator Mpa that associates with the 20S proteasome core. A pupylation-independent proteasome degradation pathway has recently been discovered that is mediated by the ATP-independent bacterial proteasome activator Bpa (also referred to as PafE), and that appears to play a role under stress conditions. In this review, mechanistic principles of bacterial proteasomal degradation are discussed and compared with functionally related elements of the eukaryotic ubiquitin-proteasome system. Special attention is given to an understanding on the molecular level based on structural and biochemical analysis. Wherever available, discussion of in vivo studies is included to highlight the biological significance of this unusual bacterial degradation pathway.

BACKGROUND Resistance to clarithromycin in Helicobacter pylori (H pylori) is mediated by mutations in the domain V of the 23S rRNA gene (A2142G, A2143G, A2142C). Other polymorphisms in the 23S rRNA gene have been reported to cause low-level clarithromycin resistance but their importance is still under debate. In this study, we aimed to develop and evaluate the CRHP Finder webtool for detection of the most common mutations mediating clarithromycin resistance from whole-genome sequencing (WGS) data. Moreover, we included an analysis of 23 H pylori strains from Danish patients between January 2017 and September 2019 in Copenhagen, Denmark. MATERIALS AND METHODS The CRHP Finder detects the fraction of each of the four nucleotides in nucleotide positions 2142, 2143, 2182, 2244 and 2712 of the 23S rRNA gene in H pylori (E coli numbering) by aligning raw sequencing reads (fastq format) with k-mer alignment (KMA). The nucleotide distribution in each position is compared to previously described point mutations mediating clarithromycin resistance in H pylori, and a genotypic prediction of the clarithromycin resistance phenotype is presented as output. For validation of the CRHP webtool, 137 fastq paired-end sequencing datasets originating from a well-characterized strain collection of H pylori were analyzed. RESULTS The CRHP Finder correctly identified all resistance mutations reported in the sequencing data of 137 H pylori strains. In the 23 Danish H pylori strains, CRHP Finder detected A2143G (13%) in all resistant strains, and T2182C (13%) and C2244T (4,3%) nucleotide exchanges in only susceptible strains. CONCLUSION In this study, we present the validation of the first webtool for H pylori resistance prediction based on the detection of 23S rRNA mutations (A2142C, A2142G, A2143G, T2182C, C2244T, T2712C) from WGS data of H pylori.