19The COVID-19 outbreak has had a major impact on clinical microbiology laboratories in the past 20 several months. This commentary covers current issues and challenges for the laboratory 21 diagnosis of infections caused by SARS-CoV-2. In the pre-analytical stage, collecting the proper 22 respiratory tract specimen at the right time from the right anatomic site is essential for a 23 prompt and accurate molecular diagnosis of COVID-19. Appropriate measures are required to 24 keep laboratory staff safe while producing reliable test results. In the analytic stage, real-time 25 RT-PCR assays remain the molecular test of choice for the etiologic diagnosis of SARS-CoV-2 26 infection while antibody-based techniques are being introduced as supplemental tools. In the 27 postanalytical stage, testing results should be carefully interpreted using both molecular and 28 serological findings. Finally, random access, integrated devices available at the point of care 29 with scalable capacities will facilitate the rapid and accurate diagnosis and monitoring of SARS-30 CoV-2 infections and greatly assist in the control of this outbreak. 31 32

Methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pyogenes (group A streptococcus [GrAS]) cause serious and sometimes fatal human diseases. They are among the many Gram-positive pathogens for which resistance to leading antibiotics has emerged. As a result, alternative therapies need to be developed to combat these pathogens. We have identified a novel bacteriophage lysin (PlySs2), derived from a Streptococcus suis phage, with broad lytic activity against MRSA, vancomycin-intermediate S. aureus (VISA), Streptococcus suis, Listeria, Staphylococcus simulans, Staphylococcus epidermidis, Streptococcus equi, Streptococcus agalactiae (group B streptococcus [GBS]), S. pyogenes, Streptococcus sanguinis, group G streptococci (GGS), group E streptococci (GES), and Streptococcus pneumoniae. PlySs2 has an N-terminal cysteine-histidine aminopeptidase (CHAP) catalytic domain and a C-terminal SH3b binding domain. It is stable at 50°C for 30 min, 37°C for >24 h, 4°C for 15 days, and ؊80°C for >7 months; it maintained full activity after 10 freeze-thaw cycles. PlySs2 at 128 g/ml in vitro reduced MRSA and S. pyogenes growth by 5 logs and 3 logs within 1 h, respectively, and exhibited a MIC of 16 g/ml for MRSA. A single, 2-mg dose of PlySs2 protected 92% (22/24) of the mice in a bacteremia model of mixed MRSA and S. pyogenes infection. Serially increasing exposure of MRSA and S. pyogenes to PlySs2 or mupirocin resulted in no observed resistance to PlySs2 and resistance to mupirocin. To date, no other lysin has shown such notable broad lytic activity, stability, and efficacy against multiple, leading, human bacterial pathogens; as such, PlySs2 has all the characteristics to be an effective therapeutic.

GATA-1 and the ets factor PU.1 have been reported to functionally antagonize one another in the regulation of erythroid versus myeloid gene transcription and development. The CCAAT enhancer binding protein ⑀ (C/EBP⑀) is expressed as multiple isoforms and has been shown to be essential to myeloid (granulocyte) terminal differentiation. We have defined a novel synergistic, as opposed to antagonistic, combinatorial interaction between GATA-1 and PU.1, and a unique repressor role for certain C/EBP⑀ isoforms in the transcriptional regulation of a model eosinophil granulocyte gene, the major basic protein (MBP). The eosinophil-specific P2 promoter of the MBP gene contains GATA-1, C/EBP, and PU.1 consensus sites that bind these factors in nuclear extracts of the eosinophil myelocyte cell line, AML14.3D10. The promoter is transactivated by GATA-1 alone but is synergistically transactivated by low levels of PU.1 in the context of optimal levels of GATA-1. The C/EBP⑀ 27 isoform strongly represses GATA-1 activity and completely blocks GATA-1/PU.1 synergy. In vitro mutational analyses of the MBP-P2 promoter showed that both the GATA-1/PU.1 synergy, and repressor activity of C/EBP⑀ 27 are mediated via protein-protein interactions through the C/EBP and/or GATA-binding sites but not the PU.1 sites. Co-immunoprecipitations using lysates of AML14.3D10 eosinophils show that both C/EBP⑀ 32/30 and ⑀ 27 physically interact in vivo with PU.1 and GATA-1, demonstrating functional interactions among these factors in eosinophil progenitors. Our findings identify novel combinatorial protein-protein interactions for GATA-1, PU

BackgroundIn previous studies, Propionibacterium acnes was cultured from intervertebral disc tissue of ~25% of patients undergoing microdiscectomy, suggesting a possible link between chronic bacterial infection and disc degeneration. However, given the prominence of P. acnes as a skin commensal, such analyses often struggled to exclude the alternate possibility that these organisms represent perioperative microbiologic contamination. This investigation seeks to validate P. acnes prevalence in resected disc cultures, while providing microscopic evidence of P. acnes biofilm in the intervertebral discs.MethodsSpecimens from 368 patients undergoing microdiscectomy for disc herniation were divided into several fragments, one being homogenized, subjected to quantitative anaerobic culture, and assessed for bacterial growth, and a second fragment frozen for additional analyses. Colonies were identified by MALDI-TOF mass spectrometry and P. acnes phylotyping was conducted by multiplex PCR. For a sub-set of specimens, bacteria localization within the disc was assessed by microscopy using confocal laser scanning and FISH.ResultsBacteria were cultured from 162 discs (44%), including 119 cases (32.3%) with P. acnes. In 89 cases, P. acnes was cultured exclusively; in 30 cases, it was isolated in combination with other bacteria (primarily coagulase-negative Staphylococcus spp.) Among positive specimens, the median P. acnes bacterial burden was 350 CFU/g (12 - ~20,000 CFU/g). Thirty-eight P. acnes isolates were subjected to molecular sub-typing, identifying 4 of 6 defined phylogroups: IA1, IB, IC, and II. Eight culture-positive specimens were evaluated by fluorescence microscopy and revealed P. acnes in situ. Notably, these bacteria demonstrated a biofilm distribution within the disc matrix. P. acnes bacteria were more prevalent in males than females (39% vs. 23%, p = 0.0013).ConclusionsThis study confirms that P. acnes is prevalent in herniated disc tissue. Moreover, it provides the first visual evidence of P. acnes biofilms within such specimens, consistent with infection rather than microbiologic contamination.

BackgroundThe relationship between intervertebral disc degeneration and chronic infection by Propionibacterium acnes is controversial with contradictory evidence available in the literature. Previous studies investigating these relationships were under-powered and fraught with methodical differences; moreover, they have not taken into consideration P. acnes’ ability to form biofilms or attempted to quantitate the bioburden with regard to determining bacterial counts/genome equivalents as criteria to differentiate true infection from contamination. The aim of this prospective cross-sectional study was to determine the prevalence of P. acnes in patients undergoing lumbar disc microdiscectomy.Methods and FindingsThe sample consisted of 290 adult patients undergoing lumbar microdiscectomy for symptomatic lumbar disc herniation. An intraoperative biopsy and pre-operative clinical data were taken in all cases. One biopsy fragment was homogenized and used for quantitative anaerobic culture and a second was frozen and used for real-time PCR-based quantification of P. acnes genomes. P. acnes was identified in 115 cases (40%), coagulase-negative staphylococci in 31 cases (11%) and alpha-hemolytic streptococci in 8 cases (3%). P. acnes counts ranged from 100 to 9000 CFU/ml with a median of 400 CFU/ml. The prevalence of intervertebral discs with abundant P. acnes (≥ 1x103 CFU/ml) was 11% (39 cases). There was significant correlation between the bacterial counts obtained by culture and the number of P. acnes genomes detected by real-time PCR (r = 0.4363, p<0.0001).ConclusionsIn a large series of patients, the prevalence of discs with abundant P. acnes was 11%. We believe, disc tissue homogenization releases P. acnes from the biofilm so that they can then potentially be cultured, reducing the rate of false-negative cultures. Further, quantification study revealing significant bioburden based on both culture and real-time PCR minimize the likelihood that observed findings are due to contamination and supports the hypothesis P. acnes acts as a pathogen in these cases of degenerative disc disease.