First report of Shigella sonnei carrying a blaCTX-M-15 sexually transmitted among men who have sex with men.

Epidemiology of shigellosis has drastically changed in recent years due to globalization and sexual risk behaviors. Here, through whole-genome sequencing, we characterized two ESBL-producing Shigella sonnei strains (ShSoBUH1 and ShSoBUH2) carrying a blaCTX-M-15 among men who have sex with men (MSM), who had not recently traveled and presented sexual risk behaviors. Both strains harbored IncB/O/K/Z and IncFII plasmids, which carry aadA1, aadA5, sul1, sul2, dfrA1, dfrA17, mph(A), erm(B), tet(B), qacE and blaCTX-M-15 genes conferring resistance to 2nd and 3rd generation cephalosporins, cotrimoxazole, erythromycin, azithromycin and quinolones. IncFII plasmids containing blaCTX-M-15 from ShSoBUH1 and ShSoBUH2 presented 99,8-99,9% similarity with plasmids from another five CTX-M-15 S. sonnei strains detected in Belgium and Switzerland. A single-nucleotide polymorphism (SNP) analysis determined that the study strains differed by 361 SNPs, belonging to different clusters. To the best of our knowledge, this is the first report describing two extensively drug-resistant (XDR) CTX-M-15 S. sonnei strains in MSM.

Brain abscess caused by Porphyromonas gingivalis and Eubacterium nodatum mimicking acute stroke.

We described a case of a 65-year-old man with a brain abscess caused by Porphyromonas gingivalis and Eubacterium nodatum. The patient presented right central facial nerve palsy, mutism and right hemiparesis at the examination. The patient underwent a left frontal craniotomy with evacuation of the brain abscess. Specimens were collected for microbiological analysis and intravenous treatment was started with levetiracetam, dexamethasone, meropenem (1 g/8 h) and linezolid (600 mg/12 h). After identification of anaerobic bacteria the antibiotic treatment was changed to piperacillin/tazobactam (4 g/0,5 g/8 h), fulfilling 8 weeks of antibiotic with good clinical and radiological evolution.

Fusobacterium ulcerans: from gut to commensal and bacterial translocation?

The genus Fusobacterium contains currently 13 species presenting as non-sporing, obligate anaerobic, Gram-negative fusiform rods. Fusobacterium ulcerans was discovered in 1988 causing tropical ulcers. We present the case of a patient with diverticulitis complicated with bacteremia. Both aerobic bottles were positive at 20 and 24 h, while one anaerobic bottle was positive at 36 h. Escherichia coli and Fusobacterium ulcerans were identified from subcultures by MALDI-TOF MS with a score of 2.02 and 2.35, respectively. The 16S rRNA gene was sequenced in order to confirm the identification of F. ulcerans with a 100% homology to the reference strain. The patient was treated with 4 g/0,5 g of IV piperacillin/tazobactam and later with 1 g/0,2 g of amoxicillin/clavulanic during 7 days with good clinical evolution.

Is Bacteroides finegoldii a new bacterial pathogen?

Bacteoides finegoldii is a Gram-negative, rod-shaped and obligate anaerobic bacteria isolated in human feces during studies of intestinal microbiota. We present a case report in which B. finegoldii was isolated and identified from the blood culture of a 93-year-old patient with abdominal pain. Microbiological identification was performed by MALDI-TOF MS and confirmed later by 16S rRNA sequencing. An abdomino-pelvic CT scan was conducted, showing a mass of neoplastic appearance that infiltrated the sigmoid colon and bladder, probably producing a colo-vesical fistula. Up to now, this is the first report of B. finegoldii causing human infection.

Description of eight human infections caused by Bacteroides pyogenes in a tertiary hospital of northern Spain.

Bacteroides pyogenes is a Gram-negative obligate anaerobe, saccharolytic, non-motile, non-pigment-producing and non-spore-forming rod. Reports of human infections caused by B. pyogenes are scarce, with approximately 30 cases documented in scientific literature. The aim of this study was to describe the clinical characteristics of 8 different patients and to study the in vitro antibiotic susceptibility of the strains, as well as to evaluate the in vivo activity of the prescribed treatments. We conducted a descriptive retrospective study by searching all B. pyogenes isolates from January 2010 to March 2023 at the Basurto University Hospital. This included all cases, both in monomicrobial or polymicrobial cultures. Among the eight patients, three experienced severe infections such as bacteremia and osteomyelitis. All the strains were susceptible to amoxicillin/clavulanic acid, piperacillin/tazobactam, imipenem, meropenem, clindamycin, metronidazole and moxifloxacin.

Misidentification of the SARS-CoV-2 Mu variant using commercial mutation screening assays.

Detection of mutations by multiplex real-time RT-PCR is a widely used method for the screening of SARS-CoV-2 variants, but this method has several limitations. We describe three cases in which a Mu strain containing the mutation K417N was initially misclassified as the Beta variant. We recommend the detection of P681H to distinguish between these two variants. Our experience highlights the importance of keeping track of new variants and mutations in order to adapt the current workflows.

Repurposing Positive SARS-CoV-2 Antigen Test Devices for Variant Tracking.

From the very beginning of the SARS-CoV-2 pandemic, one of the very few common opinions was that to control the expansion of the virus as many as the possible test had to be done. Antigen tests, being affordable and easy and fast to use, represented a great opportunity to expand the testing capacities of many healthcare systems. However, in 2021 with the appearance of the new SARS-CoV-2 variants, variant tracking strategies had to be implemented, which often included needing a second test to determine the variant of the patients diagnosed with antigen tests or not taking these samples into consideration at all. Therefore, we proposed recovering the positive antigen test devices to include them in our routine variant tracking strategy. The recovered positive antigen test devices obtained from 1st April 2021 to 15the January 2022 were analysed following the variant tracking protocol in force. The results obtained were compared to the positive samples detected by RT-PCR which were processed for variant tracking during the same period. 21,304 samples were processed, 6297 from the recovered positive antigen devices and 15,007 from the standard nasopharyngeal swabs. Only 773 (3.63%) samples were no conclusive, 104 (1.65%) from the recovered antigen devices and 669 (4.46%) from the RT-PCR positive group. This difference was statistically significant (p < 0.01). Taking this into account the proposed method is suitable and very recommendable, as it is an important measure to have a better and immediate picture of the circulating variants in every community.

Ultrafast detection of ß-lactamase resistance in Klebsiella pneumoniae from blood culture by nanopore sequencing.

Aim: This study aimed to assess the ultra-fast method using MinION™ sequencing for rapid identification of ß-lactamase-producing Klebsiella pneumoniae clinical isolates from positive blood cultures. Methods: Spiked-blood positive blood cultures were extracted using the ultra-fast method and automated DNA extraction for MinION sequencing. Raw reads were analyzed for ß-lactamase resistance genes. Multilocus sequence typing and ß-lactamase variant characterization were performed after assembly. Results: The ultra-fast method identified clinically relevant ß-lactamase resistance genes in less than 1 h. Multilocus sequence typing and ß-lactamase variant characterization required 3-6 h. Sequencing quality showed no direct correlation with pore number or DNA concentration. Conclusion: Nanopore sequencing, specifically the ultra-fast method, is promising for the rapid diagnosis of bloodstream infections, facilitating timely identification of multidrug-resistant bacteria in clinical samples.

Klebsiella pneumoniae is a bacterium that can cause infections in the blood. These infections can be severe, especially if K. pneumoniae is not susceptible to antibiotics ('antibiotic resistant'). Tools that can detect this resistance are important. In this study, we tested one such tool called MinION™ with blood samples. In 1 h, we were able to identify the bacteria within the sample and their resistance. This type of testing would help clinicians to give the best treatment to patients. More studies are needed to prove the usefulness of MinION for processing samples from real patients.

COI vs Ct: comparing automated antigen tests cut-off index (COI) to PCR cycle threshold (Ct).

INTRODUCTION: The objective of this study was to compare the numeric cut-off index (COI) of automated antigen tests with the cycle-threshold (Ct) value. MATERIALS AND METHODS: COI values of all samples processed with the Elecsys® SARS-CoV-2 Antigen (Roche, Switzerland) from January to February 2022 were retrieved and the positive were compared to RdRP Cts of Allplex Variants I (Seegene, Korea). COI between 0.6 and 1 were considered indeterminate and overproved by RT-PCR. RESULTS: From 13,937 samples 7944(57%) were positive and 189(1.35%) indeterminate. There was a strong correlation between Cts and COI values at the positive samples, but that was not the case for indeterminates. CONCLUSIONS: COI values of the positive samples (COI >1) are comparable to Ct values and may therefore be used as proxy viral loads, however this is not the case for indeterminate samples.

Disseminated Nocardia farcinica infection associated with bacteraemia and osteomyelitis pubis in an elderly patient.

OBJECTIVE: We describe a rare case of a disseminated Nocardia farcinica infection associated with hip osteomyelitis. METHODS: A 91-year-old female patient was admitted with oedema of her right leg, fever of 38 °C and data consistent with ruptured Baker's cyst. A disseminated Nocardia farcinica infection including bloodstream infection, pneumonia and multiple abscesses along both lower limbs was observed. RESULTS: After a four-week course of 320 mg/1600 mg/12 h of intravenous trimethoprim/sulfamethoxazole and multiple chirurgic drainages the patient was discharged with oral trimethoprim/sulfamethoxazole. Nevertheless, the patient expired done month after being discharged from the hospital. CONCLUSIONS: The implementation of a combination of intravenous antibiotics and drainages resulted in an initial improvement in the patient's condition. However, despite these interventions, the patient ultimately passed away probably due to natural causes.

Use of the Ct difference between the Nucleocapside (N) and the Spike (S) or RNA-dependent RNA polymerase (RdRP) genes as a preliminary screening for SARS-CoV-2 variants with the Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay: Searching the N in Variants.

PURPOSE: With the rise of the different Variants of Concern (VOC) and Variants of Interest (VOI) in order to control the SARS-CoV-2 pandemic, strategies for accurately tracking these different variants have been developed. While most of these strategies rely heavily on specific PCRs targeting the characteristic mutations of some lineages, several approaches using the alterations at the cycle threshold (Ct) of different commercial PCR diagnostic tests have been described. The objective of this study is to analyse the use of the Ct difference at the Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Seegene, Korea) between the Nucleocapside (N) and the Spike (S) or RNA-dependent RNA polymerase (RdRP) genes as a preliminary screening for variant tracking. METHODS: The samples analysed with the Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay from 1st of March 2021 to 26th of December 2021 were selected. The Ct values for N, S, RdRP were collected, and the differences between N and S (ΔS) and N and RdRP (ΔRdRP) were calculated. Using ΔS and ΔRdRP a diagnostic test was designed and these results were compared to the routine Variant assessment. RESULTS: The mean ΔS and ΔRdRP were characteristic for Alpha and Delta. This difference was statistically significant. For Every analysed Variant the diagnostic test achieved a higher than 90% sensitivity with a noteworthy performance with the Omicron variant (97% sensitivity and 90% specificity). CONCLUSIONS: The analysis of the Ct alterations at the Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay may be a suitable method for an early approach to SARS-CoV-2 variant assessment.

Ultra-fast direct method for identifying microorganisms from BACTEC lytic/10 anaerobic/F flasks.

Background: Fast diagnosis of bloodstream infections remains the most important challenge for clinical microbiologists. The introduction of the mass-spectrometry represents a breakthrough, although several methods are already commonly used for the direct identification from positive blood cultures we present a faster method (ultra fast) for Lytic anaerobic flasks. Methods: We compare the ultra-fast (UF) method with the extensively employed differential centrifugation method (DC) and both to routine identification after 18-24 h of incubation. UF and DC method correlation rates to the gold standard were calculated, and statistical significance was proved with the Z test. Results: UF performed better overall than DC, with this difference being statistically significant. This tendency was observed in every subanalysis.

First Report of New Delhi Metallo-ß-Lactamase-6 (NDM-6) in a Clinical Acinetobacter baumannii Isolate From Northern Spain.

The objective of this study was the phenotypic and genotypic characterization of a carbapenem resistant Acinetobacter baumannii (CRAB) isolate. The isolate, recovered in Northern Spain in 2019, was identified by MALDI-TOF to the species level. Antimicrobial susceptibility testing was performed using the Phoenix BD NMIC-502 Panel, E-test, and broth microdilution methods. The presence of a metallo-ß-lactamase (MBL) was verified by PCR and immunochromatographic assays. The genetic location of the MBL was confirmed using S1-pulsed-field gel electrophoresis (S1-PFGE) followed by Southern blot hybridization. Whole genome sequencing (WGS) was completed using the Miseq and MinION platforms, followed by core-genome MLST (cgMLST) and seven-locus MLST analysis. The CRAB was assigned ST85 (Pasteur scheme) and ST957 (Oxford scheme) representing international clone (IC) 9 and harbored the intrinsic ß-lactamase OXA-94 with ISAba1 upstream of it, and the MBL bla NDM-6. Hybridization experiments revealed that the bla NDM-6 was encoded on the chromosome. Using WGS the bla NDM-6 environment could be identified arranged in the following order: ISAba14, aphA6, ISAba125, bla NDM-6, ble MBL, trpF, dsbC, cutA, and ISAba14. Downstream, a 10,462 bp duplication was identified, including a second copy of bla NDM-6 in the following genetic composition: ISAba125, bla NDM-6, ble MBL, trpF, dsbC, cutA, and ISAba14. To our knowledge, this is the first description of bla NDM-6 in A. baumannii. The MBL was present in two copies in the chromosome in a new genetic environment associated with IS elements highlighting the contribution of mobile genetic elements in the dissemination of this gene.