Bloodstream infections caused by Magnusiomyces capitatus and Magnusiomyces clavatus: epidemiological, clinical and microbiological features of two emerging yeast species

Background: Magnusiomyces clavatus and Magnusiomyces capitatus are emerging yeasts with intrinsic resistance to many commonly used antifungal agents. Identification is difficult, and determination of susceptibility patterns with commercial and reference methods is equally challenging. For this reason, few data on invasive infections by Magnusiomyces spp. are available. Objectives: To determine the epidemiology and susceptibility of Magnusiomyces isolates from bloodstream infections (BSI) isolated in Germany and Austria from 2001-2020. Methods: In seven institutions a total of 34 Magnusiomyces BSI were identified. Identification was done by ITS sequencing and MALDI-TOF MS. Antifungal susceptibility was determined by EUCAST broth microdilution and gradient tests. Results: Of the 34 isolates, M. clavatus was more common (N=24) compared to M. capitatus (N=10). BSI by Magnusiomyces spp. were more common in men (62%) and mostly occurred in patients with haemato-oncological malignancies (79%). The highest in vitro antifungal activity against M. clavatus / M. capitatus was observed for voriconazole (MIC 50 0.03/0.125 mg/L), followed by posaconazole (MIC 50 0.125/0.25 mg/L). M. clavatus isolates showed overall lower MICs compared to M. capitatus . With the exception of amphotericin B, low essential agreement between gradient test and microdilution was recorded for all antifungals (0-70%). Both species showed distinct morphologic traits on ChromAgar Orientation and Columbia blood agar, which can be used for differentiation if no MALDI-TOF or molecular identification is available. Conclusion: Most BSI were caused by M. clavatus. The lowest MICs were recorded for voriconazole. Gradient tests demonstrated unacceptably low agreement and should preferably not be used for susceptibility testing of Magnusiomyces spp.

Multiple Antimicrobial Resistance in Methicillin-Resistant Staphylococcus sciuri Group Isolates from Wild Ungulates in Spain

The aim of this study was to investigate the presence of methicillin-resistant Staphylococcus (MRS) strains in non-managed wild ungulates present in a typical Mediterranean forest in Spain. For this purpose, nasal swabs were obtained from 139 animals: 90 wild boar (Sus scrofa), 42 red deer (Cervus elaphus) and 7 fallow deer (Dama dama), which were subsequently pre-enriched in BHI+ NaCl (6.5%) (24 h/37 °C), and then seeded in Columbia blood agar (24 h/37 °C)). The presence of the mecA gene was investigated by PCR, first from the confluent and then from individual colonies. A total of 10 mecA+ colonies were obtained of which only seven showed phenotypic resistance to oxacillin/cefoxitin (methicillin resistance). All MRS strains belonged to the Staphylococcus sciuri group. Methicillin-resistant Staphylococcus aureus (MRSA) was not detected. In addition, a significant number of MRS strains showed resistance to other antimicrobials, mainly β-lactam (7/7), gentamicin (7/7), fusidic acid (6/7) and quinupristin–dalfopristin (6/7), showing an irregular correlation with their coding genes. The genetic profiles grouped the seven strains obtained according to the bacterial species but not in relation to the animal source or the geographical place of origin. The presence of SCCmec type III, common to animals and humans, has been detected in three of the strains obtained. In conclusion, the study reveals that the wild ungulates investigated play a role as potential reservoirs of multi-resistant strains of MRS. Such strains, due to their characteristics, can be easily transferred to other wild or domestic animal species and ultimately to humans through their products.

Method-Dependent Implications in Foodborne Pathogen Quantification: The Case of Campylobacter coli Survival on Meat as Comparatively Assessed by Colony Count and Viability PCR

The aim of the present study was to address method-dependent implications during the quantification of viable Campylobacter coli cells on meat over time. Traditional colony counting on selective and non-selective culture media along with an optimized viability real-time PCR utilizing propidium monoazide-quantitative PCR (PMA-qPCR), spheroplast formation and an internal sample process control (ISPC), were comparatively evaluated for monitoring the survival of C. coli on fresh lamb meat during refrigeration storage under normal atmospheric conditions. On day zero of three independent experiments, lamb meat pieces were artificially inoculated with C. coli and then stored under refrigeration for up to 8 days. Three meat samples were tested on different days and the mean counts were determined per quantification method. An overall reduction of the viable C. coli on lamb meat was observed regardless of the applied quantification scheme, but the rate of reduction followed a method-dependent pattern, the highest being observed for colony counting on modified charcoal cefoperazone deoxycholate agar (mCCDA). Univariate ANOVA indicated that the mean counts of viable C. coli using PMA-qPCR were significantly higher compared to Columbia blood agar (CBA) plating (0.32 log10 cell equivalents, p = 0.015) and significantly lower when mCCDA was compared to CBA plating (0.88 log10 CFU, p < 0.001), indicating that selective culture on mCCDA largely underestimated the number of culturable cells during the course of meat storage. PMA-qPCR outperformed the classical colony counting in terms of quantifying both the culturable and viable but non-culturable (VBNC) C. coli cells, which were generated over time on meat and are potentially infectious and equally important from a public health perspective as their culturable counterparts.

Antimicrobial susceptibility pattern of oral viridans group Streptococci in children at risk of infective endocarditis

Infective endocarditis (IE) is an important clinical disease in children with a mortality rate of 11.6%. Prophylaxis with antibiotics is one of the most commonly used methods in children at risk of IE; therefore, the evaluation of antibiotic resistance seems necessary in view of its increasing trend. This study aimed to determine the antibiotic susceptibility pattern of oral viridans group streptococci (VGS) isolated from the dental plaque of children at risk of IE. Fifty-one plaque samples were obtained from children aged 3 to 12 years old in the period from April to July 2018. Samples were obtained with sterile swabs and were transferred to the laboratory in Brain Heart Infusion (BHI) Broth. Samples were immediately cultivated on Columbia blood agar. After identifying VGS, antimicrobial susceptibility test (AST) was performed using Mueller-Hinton agar (MHA) with sheep's blood and E-test strips for selected antibiotics. The minimum inhibitory concentration (MIC) was determined for each isolate and the results were reported as sensitive, intermediate and resistant. Fifty-one VGS bacteria were isolated from children with an average age of 7.3 ± 2.5 years. The highest resistance was observed for azithromycin in 36 (70.6%) isolates and then cefazolin in 35 (68.6%) isolates. The highest susceptibility was observed for amoxicillin in 46 (90.2%) isolates. Based on the findings of this study, amoxicillin is the most effective option for prophylaxis in children. Furthermore, cefazolin should be used with caution because bacteria resistant to this antibiotic can transfer resistance genes to other bacteria.

Initial Bacterial Adhesion and Biofilm Formation on Aligner Materials

The present study aims to assess the initial bacterial adhesion and biofilm formation on different aligner materials. A total of four different aligner materials, CA-medium (CAM), copolyester (COP), Duran (DUR), Erkodur (ERK), were tested. Stimulated human saliva was obtained from six healthy volunteers. Salivary bacteria were harvested by centrifugation, and 1 mL of the salivary suspension was injected onto each sample surface for 2 h and 3 days, respectively. The samples were then washed twice with 5 mL 0.9% NaCl solution, and non-adherent bacteria were removed. The adherent microorganisms were dislodged from the sample surfaces after ultrasonication for 4 min in 1 mL 0.9% NaCl on ice. After the incubation of the adherent salivary bacteria under both aerobic and anaerobic conditions on Columbia blood agar plates at 37 °C and 5% CO2 and in anaerobic jars overnight, several dilutions thereof were used for the determination of CFUs. This protocol was applied three times, obtaining an average of nine independent measurements for each material group. Overall, the differences between the tested aligner materials as well as between the materials and controls were not of statistical significance (p > 0.05). Regarding initial bacterial attachment and biofilm formation, the tested aligner materials are comparable to enamel and metal orthodontic brackets and can be therefore considered for clinical use. The four tested aligner materials CAM, COP, DUR, ERK showed no significant differences in initial microbial attachment and biofilm formation of aerobic and anaerobic species compared to enamel and conventional brackets.

Evaluation of CARBA PAcE, a novel rapid test for detection of carbapenemase-producing Enterobacterales

Introduction. Carbapenemase-producing Enterobacterales (CPE) are an increasing threat to global health. Fast detection is crucial for patient management and outbreak control. Hypothesis/Gap statement. Recently, a new commercial colorimetric test, CARBA PAcE, was released that has not yet been scientifically evaluated. Aim. Our goals were to evaluate the performance of CARBA PAcE using a large variety of different CPE. Methodology. CARBA PAcE was challenged with 107 molecularly characterized CPE and 53 non-CPE controls. Isolates were grown on Mueller-Hinton agar (MHA); in the case of a false-negative result, isolates were additionally inoculated on Columbia blood agar (CBA) and CARBA PAcE was repeated. The test was performed according to the manufacturer’s protocol. Results. CARBA PAcE showed an overall sensitivity and specificity of 72 % [confidence interval (CI) 62–80 %] and 91 % (CI 79–97 %), respectively, when isolates were grown on MHA. With growth on CBA, detection improved (especially of metallo-β-lactamases), resulting in an extrapolated sensitivity of 89 % (CI 81–94 %) for all carbapenemases and 96 % (CI 89–99 %) for the four major carbapenemases (NDM, OXA-48-like, KPC, VIM). Conclusion. CARBA PAcE is a simple and very rapid test for the detection of CPE which performs well for the major carbapenemases when isolates are grown on CBA. Laboratories should be aware of the limitations of this assay, such as moderate sensitivity when isolates are grown on more challenging agars such as MHA and the poor detection of some rare carbapenemases (e.g. IMI, OXA-58).

The Novel CarbaLux Test for Carbapenemases and Carbapenem Deactivating AmpC Beta-Lactamases

ObjectivesTo evaluate the rapid phenotypic CarbaLux test for routine diagnostics in the medical laboratory in a proof of concept study.Methodsisolates of Gram-negative bacteria suspicious for carbapenem resistance including Enterobacterales (67), Pseudomonas (10), Acinetobacter (5), and Stenotrophomonas (1) species, collected between 2016 and 2018 from in-patients, were tested for carbapenemase activity using a novel fluorescent carbapenem. When subjected to extracted bacterial carbapenemases its fluorescence disappears. All bacteria to be tested were cultured on Columbia blood agar and few on other commercial media. MALDI TOF MS, molecular assays, automated MIC testing, and in part, agar diffusion tests served to characterize the isolates. For comparison, few selected bacteria were also investigated by prior phenotypic tests for carbapenemase detection.ResultsUnder UV light, the CarbaLux test allowed a rapid detection of 39/39 carbapenemase-producing bacteria, including 15 isolates with OXA carbapenemases (e.g., OXA-23, OXA-24/40-like OXA-48-like or OXA-181). Several isolates had low MICs but still expressed carbapenemases. Among Enterobacter spp., it detected six strains with hyper-produced AmpC beta-lactamases, which deactivated carbapenems but were not detectable by prior rapid phenotypic assays. An unexpected high carbapenemase activity appeared with these enzymes. They were identified as AmpC variants by inhibition with cloxacillin.ConclusionOther than prior rapid phenotypic assessments for carbapenemases, which use secondary effects such as a change of pH, the inactivation of the fluorescent carbapenem substrate can be visualized directly under UV light. The new test works at 100 to 200-fold lower, therapy-like substrate concentrations. It takes advantage of the high substrate affinity to carbapenemases allowing also the detection of less reactive resistance enzymes via a trapping mechanism, even from bacteria, which might appear unsuspicious from initial antibiograms. The novel fluorescence method allows simple and safe handling, reliable readings, and documentation and is suitable for primary testing in the clinical laboratory.

Vaginal Microbiota Is Stable throughout the Estrous Cycle in Arabian Mares

Lactic acid bacteria (LAB) dominate human vaginal microbiota and inhibit pathogen proliferation. In other mammals, LAB do not dominate vaginal microbiota, however shifts of dominant microorganisms occur during ovarian cycle. The study objectives were to characterize equine vaginal microbiota in mares by culture-dependent and independent methods and to describe its variation in estrus and diestrus. Vaginal swabs from 8 healthy adult Arabian mares were obtained in estrus and diestrus. For culture-dependent processing, bacteria were isolated on Columbia blood agar (BA) and Man Rogosa Sharpe (MRS) agar. LAB comprised only 2% of total bacterial isolates and were not related to ovarian phases. For culture-independent processing, V3/V4 variable regions of the 16S ribosomal RNA gene were amplified and sequenced using Illumina Miseq. The diversity and composition of the vaginal microbiota did not change during the estrous cycle. Core equine vaginal microbiome consisted of Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria at the phylum level. At the genus level it was defined by Porphyromonas, Campylobacter, Arcanobacterium, Corynebacterium, Streptococcus, Fusobacterium, uncultured Kiritimatiaellae and Akkermansia. Lactobacillus comprised only 0.18% of the taxonomic composition in estrus and 0.37% in diestrus. No differences in the relative abundance of the most abundant phylum or genera were observed between estrus and diestrus samples.

Co-Culture of Acinetobacter calcoaceticus-baumannii complex and Staphylococcus saprophyticus Supports Simple Point Contamination Model in Recent Cases of Transfusion-Related Sepsis

The CDC recently reported a series of four septic transfusion reactions across three states resulting from contamination of apheresis platelet products. The apheresis platelets were contaminated with strains of both Acinetobacter calcoaceticus-baumannii complex (ACBC) and Staphylococcus saprophyticus (Ss). Two of the reported septic transfusion reactions occurred at our institution. The CDC investigation showed that isolates of each species were genetically related and suggested a point source of contamination. However, the contamination of blood products with ACBC is rare and the co-occurrence of these two species in all four cases was unusual. We hypothesized that there was an augmentative interaction between the clinical isolates of ACBC and Ss from these cases that contributed to their repeated co-occurrence. To test this hypothesis, we compared the growth characteristics of ACBC and Ss when cultured together versus independently. We used isolates from the contaminated platelets for our studies and performed experiments using both solid and liquid growth media. Experiments on solid media assessed density of growth and macroscopic morphology after cross-streaking on Columbia Blood Agar (CBA) and Luria-Bertani (LB) agar. Experiments in liquid media assessed growth by CFU counts in LB broth, platelet-poor plasma, and apheresis platelets. Growth in apheresis platelets was performed using standard, room temperature blood bank platelet storage conditions. Results of these experiments showed a higher CFU concentration of Ss when co-cultured with ACBC in LB broth only after several days, as compared to Ss alone under the same conditions. Otherwise, there was no evidence of augmented growth by either CFU concentration or growth rate in LB broth, plasma, or platelets at other time points. Similarly, there was no evidence of augmented growth by colony density or morphology when cross-streaking strains on either CBA or LB agar. As a result, we conclude that the co-occurrence of these two species in platelets is likely a coincidence of the point contamination suggested by the CDC investigation and not the result of growth augmentation between the two species.

Algal Lymphadenitis in a Dog Caused by Scenedesmus Species

A 6-year-old, spayed female Labrador/Weimaraner cross-breed dog that had previously lived in Arizona presented in Montana for an annual examination with an incidentally enlarged popliteal lymph node, which was subsequently biopsied. Histologically, the lymph node was expanded by eosinophil-rich granulomas with both extracellular and intrahistiocytic green algae. These algae had intracytoplasmic, birefringent, and refractile granules; readily formed 2 to 3 mm green colonies on Columbia blood agar medium; and ultrastructurally had a multilayered cell wall and intracytoplasmic chloroplasts. Amplified product from the internal transcribed spacer and D1/D2 regions of the 28S ribosomal RNA gene had high sequence identity to Scenedesmus sp. Despite similar infection in the retropharyngeal lymph node 1 year later, the animal remained otherwise healthy with no clinical signs. To the authors’ knowledge, this is the first case of Scenedesmus species infection in a dog and is a differential diagnosis for Coccidioides immitis.