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The dynamics related to the loss of stx genes from Shiga toxin-producing Escherichia coli remain unclear. Current diagnostic procedures have shortcomings in the detection and identification of STEC. This is partly owing to the fact that stx genes may be lost during an infection or in the laboratory. The aim of the present study was to provide new insight into in vivo and in vitro stx loss in order to improve diagnostic procedures. Results from the study support the theory that loss of stx is a strain-related phenomenon and not induced by patient factors. It was observed that one strain could lose stx both in vivo and in vitro. Whole genome comparison of stx-positive and stx-negative isolates from the same patient revealed that different genomic rearrangements, such as complete or partial loss of the parent prophage, may be factors in the loss of stx. Of diagnostic interest, it was shown that patients can be co-infected with different E. coli pathotypes. Therefore, identification of eae-positive, but stx-negative isolates should not be interpreted as “Shiga toxin-lost” E. coli without further testing. Growth and recovery of STEC were supported by different selective agar media for different strains, arguing for inclusion of several media in STEC diagnostics.

Streptococcus salivarius (S. salivarius) is a prominent oral commensal bacterium with probiotic potential and relevance to both oral and systemic health. Accurate and accessible methods for quantitative measurement of this species are needed to support microbiota studies and clinical interventions. We describe a simple, low-cost, culture-based method for quantifying S. salivarius in human saliva using Mitis-Salivarius Agar. Saliva samples from 18 healthy adult volunteers were analyzed through serial dilutions and selective plating. CFU (colony forming unit)/mL were calculated after 24 and 48 h incubation. The method proved reliable for quantifying S. salivarius in concentrations ranging from 5.8 × 105 to 6.1 × 108 CFU/mL. Although Mitis-Salivarius Agar is a standard medium, we demonstrate its systematic validation and optimization for human saliva in a low-resource clinical setting, where molecular tools are often unavailable.

Epidemiological data show that the composition of gut microbiota influences host health, disease status, and even behaviour. However, to confirm these epidemiological observations in controlled experiments, pure cultures of gut anaerobes must be obtained. Since the culture of gut anaerobes is not a simple task due to the large number of bacterial species colonising the intestinal tract, in this study we inoculated 174 different culture media with caecal content from adult hens, and compared the microbiota composition in the original caecal samples and in bacterial masses growing in vitro by 16S rRNA sequencing. In total, 42% of gut microbiota members could be grown in vitro and since there were some species which were not cultured but for which the culture conditions are known, it is likely that more than half of chicken gut microbiota can be grown in vitro. However, there were two lineages of Clostridiales and a single lineage of Bacteroidetes which were common in chicken caecal microbiota but resistant to culture. Of the most selective culture conditions, nutrient broths supplemented with mono- or di-saccharides, including those present in fruits, positively selected for Lactobacillaceae. The addition of bile salts selected for Veillonellaceae and YCFA (yeast casitone fatty acid agar) enriched for Desulfovibrionaceae. In addition, Erysipelotrichaceae were positively selected by colistin, trimethoprim, streptomycin and nalidixic acid. Culture conditions tested in this study can be used for the selective enrichment of desired bacterial species but also point towards the specific functions of individual gut microbiota members.

Entomopathogenic fungi (EPF) in Croatian forests are known only from observations of insect cadavers that show obvious signs of disease. To date, their presence in soils has not been investigated. The aim of this study was to investigate their occurrence, diversity, and distribution, and to assess their density in tested soils. Soil samples were collected during 2018, 2019, and 2020 at different localities throughout the country, and analyzed by using a method of isolation of fungi on selective culture media. To assess the density of EPF in tested soils, colonies of individual fungal species were counted and recorded; the results were expressed as the number of colony-forming units (CFU) per gram of dry soil. After morphological and molecular analysis, five entomopathogenic fungal genera were identified: Beauveria spp., Metarhizium spp., Purpureocillium spp., Lecanicillium spp., and Paecilomyces spp. Results also showed that the range of a total EPF colony density in the soil varies from 4 × 103 to 27.4 × 103 CFU g−1. The most common were EPF of the genus Beauveria, which were recorded at four of five locations, and at 16 of 25 sampling points, but the highest average number (density) of colonies belonged to the genus Metarhizium. Since this type of research was never conducted in Croatia previously, this is the first evidence that insect pathogenic fungi are present in soils of different natural forest habitats. Such research can be useful in selecting and utilizing entomopathogens that are suitable for biological pest control in certain target areas.

Abstract Bacteriological agar plates are commonly used to carry out experiments for the selective growth of microorganisms and the isolation of single-strain colonies. However, the presence of agar itself may be a confounding factor since it may serve as a source of carbon and energy. Moreover, there have been ongoing constraints on the production and sourcing of agar. These concerns have led to an interest in the development of agar substitutes. Silica hydrogels are entirely inorganic carbon-free polymeric materials that lack any source of micronutrients. Herein, a revised method for the preparation of silica hydrogels as a solid culture medium is reported. These gels can be formulated with a range of nutrient-rich or minimal media supplemented with various carbon sources, and can be manipulated in the same manner as agar gels. Their use for the culture and isolation of diverse microorganisms, including both Gram-positive and Gram-negative bacteria, yeast, and filamentous fungi is demonstrated. These silica hydrogels supplemented with either antibiotics or other molecules of interest can also be used for microbial selection experiments. Silica hydrogel solid culture media as a carbon-free alternative to agar.

Purpose of ReviewVentilator-associated pneumonia (VAP) is one of the most common infections in the ICU. Prompt diagnosis is vital as mortality increases with delayed antibiotic therapy. However, accurate diagnosis is challenging due to non-specific clinical features in a complicated patient cohort. Microbiological culture data remains a crucial aspect in confirming diagnosis.Recent FindingsLiterature data comparing the benefit of invasive respiratory sampling to non-invasive is inconclusive. Differences in culturing practices translate in overidentification of organisms of unclear significance. Positive culture data in a low pre-test probability does not differentiate between true infection and colonization resulting in overtreatment. Furthermore, there are also opportunities for modifying the reporting of respiratory tract cultures that can better guide antimicrobial therapy.SummaryUnder the umbrella of antimicrobial stewardship, diagnostic stewardship can be incorporated to create a systematic approach that would target culturing practices to match the right pre-test probability. Ideal outcome will be targeting cultures to the right patient population and minimizing unnecessary treatment.

Malassezia species are fastidious and slow-growing yeasts in which isolation from polymicrobial samples is hampered by fast-growing microorganisms. Malassezia selective culture media are needed. Although cycloheximide is often used, some fungi, including the chief human commensal Candida albicans, are resistant to this compound. This study aimed to test whether the macrolide rapamycin could be used in combination with cycloheximide to develop a Malassezia-selective culture medium. Rapamycin susceptibility testing was performed via microdilution assays in modified Dixon against two M. furfur and five Candida spp. The MIC was the lowest concentration that reduced growth by a minimum of 90%. Rapamycin ± cycloheximide 500 mg/L was also added to FastFung solid, and yeast suspensions were inoculated and incubated for 72 h. Rapamycin MICs for Candida spp. ranged from 0.5 to 2 mg/L, except for C. krusei, for which the MIC was >32 mg/L. M. furfur stains were rapamycin-resistant. Rapamycin and cycloheximide supplementation of the FastFung medium effectively inhibited the growth of non-Malassezia yeast, including cycloheximide-resistant C. albicans and C. tropicalis. Based on our findings, this “MalaSelect” medium should be further evaluated on polymicrobial samples for Malassezia isolation and culture.

Mediterranean pine engraver, Orthotomicus erosus was never considered as a significant pest in Croatia and did not appear in high population densities until 2017, when it reached outbreak level in Aleppo pine stands. The beetle was first detected in Marjan Forest Park, Split, and was soon recorded in other parts of the Dalmatian coast. Soon after the outbreak occurred, we observed that all of the attacked trees exhibit severe blue staining in the sapwood which indicated fungal infection caused by the Ophiostomatales group of fungi. This raised the need to investigate their relationship with O. erosus and the pine decline, and the main aim of this study was to isolate and identify them. Isolates were obtained from adult O. erosus beetles, their galleries, and blue-stained sapwood, and identified according to the morphological characteristics and DNA sequencing. A total of six Ophiostomatales (Ophiostoma ips, O. piceae, Graphilbum cf. rectangulosporium, O. floccosum, Sporothrix pseudoabietina and Ceratocystiopsis cf. minuta) were identified in the study. This is the first record of Ophiostomatales as organisms associated with the pest O. erosus and pine species in Croatia.

Background Identification of carbapenemase-producing Enterobacteriaceae (CPE) in faecal specimens is challenging. This fact is particularly critical because low-level carbapenem-resistant organisms such as IMP-producing CPE are most prevalent in Japan. We developed a modified selective medium more suitable for IMP-type CPE. Methods Fifteen reference CPE strains producing different types of β-lactamases were used to evaluate the commercially available CHROMagar KPC and chromID CARBA as well as the newly prepared MC-ECC medium (CHROMagar ECC supplemented with meropenem, cloxacillin, and ZnSO 4 ) and M-ECC medium (CHROMagar ECC supplemented with meropenem and ZnSO 4 ). A total of 1035 clinical samples were then examined to detect CPE using chromID CARBA and M-ECC medium. Results All tested strains producing NDM-, KPC-, and OXA-48-carbapenemases were successfully cultured in the media employed. Although most of the IMP-positive strains did not grow in CHROMagar KPC, chromID CARBA, or MC-ECC, all tested strains grew on M-ECC. When faecal samples were applied to the media, M-ECC medium allowed the best growth of IMP-type CPE with a significantly higher sensitivity (99.3%) than that of chromID CARBA (13.9%). Conclusions M-ECC medium was determined as the most favourable selective medium for the detection of IMP-type CPE as well as other types of CPE.

Yeast species of sound and sour rot-damaged grapes were analysed during fermentation and grape ripening in the vineyard, using general and selective culture media. During 2003 and 2004 vintages, microvinifications were carried out with sound grapes to which different amounts of grapes with sour rot were added. The wine spoilage species Zygosaccharomyces bailii was only recovered during fermentations with sour rot, reaching 5.00 log CFUmL 1 (2003) and 2.48 log CFUmL 1 (2004) at the end of fermentation. The study of yeast populations during the sour rot ripening process (2005 vintage) showed that the veraisondamaged grapes always exhibited higher total yeast counts and a much greater diversity of species. From a total of 22 ascomycetous species, 17 were present only in damaged grapes. The most frequent species were Issatchenkia occidentalis and Zygoascus hellenicus. The spoilage species Z. bailii and Zygosaccharomyces bisporus were consistently isolated exclusively from damaged grapes. This work demonstrates that one of the most dangerous wine spoilage species, Z. bailii, is strongly associated with sour rot grapes and survives during fermentation with Saccharomyces cerevisiae. The use of selective media provides a more accurate characterization of grape contamination species.