Gonococcal mastitis. / Gonokokkmastitt.

A young woman experienced pain and swelling in a non-lactating breast. The culture test result showed an unusual microbe, which is increasingly prevalent in Norway and internationally.

Microbiological diagnosis of pleural infections: a comparative evaluation of a novel syndromic real-time PCR panel.

Current microbial diagnostics for pleural infections are insufficient. Studies using 16S targeted next-generation sequencing report that only 10%-16% of bacteria present are cultured and that 50%-78% of pleural fluids containing relevant microbial DNA remain culture negative. As a rapid diagnostic alternative suitable for clinical laboratories, we wanted to explore a PCR-based approach. Based on the identification of key pathogens, we developed a syndromic PCR panel for community-acquired pleural infections (CAPIs). This was a pragmatic PCR panel, meaning that it was not designed for detecting all possibly involved bacterial species but for confirming the diagnosis of CAPI, and for detecting bacteria that might influence choice of antimicrobial treatment. We evaluated the PCR panel on 109 confirmed CAPIs previously characterized using culture and 16S targeted next-generation sequencing. The PCR secured the diagnosis of CAPI in 107/109 (98.2%) and detected all present pathogens in 69/109 (63.3%). Culture secured the diagnosis in 54/109 (49.5%) and detected all pathogens in 31/109 (28.4%). Corresponding results for 16S targeted next-generation sequencing were 109/109 (100%) and 98/109 (89.9%). For bacterial species included in the PCR panel, PCR had a sensitivity of 99.5% (184/185), culture of 21.6% (40/185), and 16S targeted next-generation sequencing of 92.4% (171/185). None of the bacterial species present not covered by the PCR panel were judged to impact antimicrobial therapy. A syndromic PCR panel represents a rapid and sensitive alternative to current diagnostic approaches for the microbiological diagnosis of CAPI.IMPORTANCEPleural empyema is a severe infection with high mortality and increasing incidence. Long hospital admissions and long courses of antimicrobial treatment drive healthcare and ecological costs. Current methods for microbiological diagnostics of pleural infections are inadequate. Recent studies using 16S targeted next-generation sequencing as a reference standard find culture to recover only 10%-16% of bacteria present and that 50%-78% of samples containing relevant bacterial DNA remain culture negative. To confirm the diagnosis of pleural infection and define optimal antimicrobial therapy while limiting unnecessary use of broad-spectrum antibiotics, there is a need for rapid and sensitive diagnostic approaches. PCR is a rapid method well suited for clinical laboratories. In this paper we show that a novel syndromic PCR panel can secure the diagnosis of pleural infection and detect all bacteria relevant for choice of antimicrobial treatment with a high sensitivity.

Pleural Empyema Caused by Streptococcus intermedius and Fusobacterium nucleatum: A Distinct Entity of Pleural Infections.

BACKGROUND: Many community-acquired pleural infections are caused by facultative and anaerobic bacteria from the human oral microbiota. The epidemiology, clinical characteristics, pathogenesis, and etiology of such infections are little studied. The aim of the present prospective multicenter cohort study was to provide a thorough microbiological and clinical characterization of such oral-type pleural infections and to improve our understanding of the underlying etiology and associated risk factors. METHODS: Over a 2-year period, we included 77 patients with community-acquired pleural infection, whereof 63 (82%) represented oral-type pleural infections. Clinical and anamnestic data were systematically collected, and patients were offered a dental assessment by an oral surgeon. Microbial characterizations were done using next-generation sequencing. Obtained bacterial profiles were compared with microbiology data from previous investigations on odontogenic infections, bacteremia after extraction of infected teeth, and community-acquired brain abscesses. RESULTS: From the oral-type pleural infections, we made 267 bacterial identifications representing 89 different species. Streptococcus intermedius and/or Fusobacterium nucleatum were identified as a dominant component in all infections. We found a high prevalence of dental infections among patients with oral-type pleural infection and demonstrate substantial similarities between the microbiology of such pleural infections and that of odontogenic infections, odontogenic bacteremia, and community-acquired brain abscesses. CONCLUSIONS: Oral-type pleural infection is the most common type of community-acquired pleural infection. Current evidence supports hematogenous seeding of bacteria from a dental focus as the most important underlying etiology. Streptococcus intermedius and Fusobacterium nucleatum most likely represent key pathogens necessary for establishing the infection.

A tale of two STs: molecular and clinical epidemiology of MRSA t304 in Norway 2008-2016.

The purpose of this study was to investigate the epidemiological, molecular, and clinical characteristics of MRSA t304/ST8 and t304/ST6 in Norway from 2008 to 2016. Clinical and epidemiological data were collected for each case included in the study. Strains were characterized by PCR, spa typing, antimicrobial susceptibility testing, and whole genome sequencing. The overall number of cases of MRSA t304 increased from 27 in 2008 to 203 in 2016. Most MRSA t304/ST8 cases were defined as HA-MRSA (89.9%) and diagnosed in persons with Norwegian background, many of them living in nursing homes (62.3%). The number of t304/ST8 cases declined throughout the study period and it has not been reported in Norway since 2014. The increasing MRSA t304/ST6 genotype has mainly been introduced to Norway by immigration from the Middle East, but also from other parts of the world. The t304/ST6 clone is mostly classified as CA-MRSA (75.1%), does not seem to cause serious infections, is not multi-resistant, and has not yet caused outbreaks in Norway. This study provides an example of two MRSA clones with the same spa type found in different epidemiological settings. This is very unusual, but still a reminder that spa typing in some cases may have insufficient discriminatory power for surveillance of MRSA. Our results highlight the importance of active surveillance and characterization of emerging MRSA clones with high potential for spread in the community, which may potentially cause outbreaks in healthcare facilities.

Rapid discrimination of Staphylococcus epidermidis genotypes in a routine clinical microbiological laboratory using single nucleotide polymorphisms in housekeeping genes.

PURPOSE: Staphylococcus epidermidis colonies often display several morphologies and antimicrobial susceptibility patterns when cultured from device-related infections, and may represent one or multiple genotypes. Genotyping may be helpful in the clinical interpretation, but is time consuming and expensive. We wanted to establish a method for rapid discrimination of S. epidermidis genotypes for use in a routine microbiology laboratory. METHODOLOGY: A real-time PCR targeting eight discriminatory class I or II single-nucleotide polymorphisms (SNPs) in six of the seven housekeeping genes was constructed. Post PCR, high-resolution melt (HRM) analysis using EvaGreen as fluorophore discriminated amplicons based on their percentage GC content. RESULTS: In silico, 42 representative sequence types (STs), including all major MLST group and subgroup founders, were separated into 23 different cluster profiles with a Simpson's index of diversity of 0.97. By HRM-PCR, 11 commonly encountered hospital and outbreak STs were separated into eight HRM patterns. CONCLUSION: This method can rapidly establish whether S. epidermidis strains belong to different genotypes. It can be used in patients with S. epidermidis infections, as an aid in outbreak investigations and to select strains for investigation with more discriminatory methods, saving workload and costs. Results may be obtained the same day as culture results. Its strength lies mainly in indicating differences, as some STs may have the same melt profile. Changes in S. epidermidis epidemiology may warrant alterations in the inclusion of SNPs. We believe this method can reduce the threshold for performing genotyping analysis on an increasingly important nosocomial pathogen.

Methicillin-Resistant Staphylococcus aureus CC398 in Humans and Pigs in Norway: A "One Health" Perspective on Introduction and Transmission.

BACKGROUND: Emerging livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) persist in livestock populations and represent a reservoir for transmission to humans. Understanding the routes of introduction and further transmission is crucial to control this threat to human health. METHODS: All reported cases of livestock-associated MRSA (CC398) in humans and pigs in Norway between 2008 and 2014 were included. Data were collected during an extensive outbreak investigation, including contact tracing and stringent surveillance. Whole-genome sequencing of isolates from all human cases and pig farms was performed to support and expand the epidemiological findings. The national strategy furthermore included a "search-and-destroy" policy at the pig farm level. RESULTS: Three outbreak clusters were identified, including 26 pig farms, 2 slaughterhouses, and 36 humans. Primary introductions likely occurred by human transmission to 3 sow farms with secondary transmission to other pig farms, mainly through animal trade and to a lesser extent via humans or livestock trucks. All MRSA CC398 isolated from humans without an epidemiological link to the outbreaks were genetically distinct from isolates within the outbreak clusters indicating limited dissemination to the general population. CONCLUSIONS: This study identified preventable routes of MRSA CC398 introduction and transmission: human occupational exposure, trade of pigs and livestock transport vehicles. These findings are essential for keeping pig populations MRSA free and, from a "One Health" perspective, preventing pig farms from becoming reservoirs for MRSA transmission to humans.

A Silenced vanA Gene Cluster on a Transferable Plasmid Caused an Outbreak of Vancomycin-Variable Enterococci.

We report an outbreak of vancomycin-variable vanA(+) enterococci (VVE) able to escape phenotypic detection by current guidelines and demonstrate the molecular mechanisms for in vivo switching into vancomycin resistance and horizontal spread of the vanA cluster. Forty-eight vanA(+) Enterococcus faecium isolates and one Enterococcus faecalis isolate were analyzed for clonality with pulsed-field gel electrophoresis (PFGE), and their vanA gene cluster compositions were assessed by PCR and whole-genome sequencing of six isolates. The susceptible VVE strains were cultivated in brain heart infusion broth containing vancomycin at 8 µg/ml for in vitro development of resistant VVE. The transcription profiles of susceptible VVE and their resistant revertants were assessed using quantitative reverse transcription-PCR. Plasmid content was analyzed with S1 nuclease PFGE and hybridizations. Conjugative transfer of vanA was assessed by filter mating. The only genetic difference between the vanA clusters of susceptible and resistant VVE was an ISL3-family element upstream of vanHAX, which silenced vanHAX gene transcription in susceptible VVE. Furthermore, the VVE had an insertion of IS1542 between orf2 and vanR that attenuated the expression of vanHAX Growth of susceptible VVE occurred after 24 to 72 h of exposure to vancomycin due to excision of the ISL3-family element. The vanA gene cluster was located on a transferable broad-host-range plasmid also detected in outbreak isolates with different pulsotypes, including one E. faecalis isolate. Horizontally transferable silenced vanA able to escape detection and revert into resistance during vancomycin therapy represents a new challenge in the clinic. Genotypic testing of invasive vancomycin-susceptible enterococci by vanA-PCR is advised.