Ingolstadt Hospital is a secondary 1155-bed hospital in the center of Bavaria (southeastern Germany), providing health care for about 500,000 inhabitants in the planning region 10. The hospital boasts three intensive care units and intermediate care as well as a stroke unit. The hospital has four internal and various surgical facilities. At the microbiological laboratory of Ingolstadt Hospital, SDSE isolates were cultured in thioglycolate broth, on sheep blood agar (bioMérieux, Nürtingen, Germany), Schaedler agar containing 5% sheep blood (bioMérieux), and/or chocolate agar (bioMérieux). Urine samples were cultured on sheep blood agar covered with a 50 µg pipemidic acid disk (BioRad, Marnes-la-Coquette, France) to inhibit the growth of Gram-negative bacteria. Blood cultures were grown in aerobe and anaerobe blood culture bottles using the BactAlert blood culture system (bioMérieux). After bacterial growth was detected by the BactAlert system, blood samples from aerobe bottles were given on blood and chocolate agar and additionally on Schaedler agar in the case of bacterial growth in anaerobe blood culture bottles. Species identification was either determined using Vitek 2 MS (bioMérieux) or the Vitek 2 compact (bioMérieux) with appropriate Vitek 2 identification (ID) cards.
Sheep blood agar
Manufactured by bioMérieux
Sourced in France
Sheep blood agar is a culture medium used for the isolation and identification of various microorganisms. It is made by adding defibrinated sheep blood to a base agar medium. The presence of sheep blood provides essential nutrients and growth factors for the cultivation of fastidious bacteria, as well as the ability to detect hemolytic activity, which is a useful characteristic for the differentiation of certain bacterial species.
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Lab products found in correlation
Vitek 2 compact, by bioMérieux (2 mentions)
Schaedler agar, by bioMérieux (2 mentions)
Chocolate agar, by bioMérieux (2 mentions)
Macconkey agar, by bioMérieux (2 mentions)
Bact alert blood culture system, by bioMérieux (1 mentions)
Vitek 2 ms, by bioMérieux (1 mentions)
Bactec 9050, by BD (1 mentions)
Bacto yeast extract, by BD (1 mentions)
Brain heart infusion, by BD (1 mentions)
Proteose peptone, by Thermo Fisher Scientific (1 mentions)
Brain heart infusion broth bhi, by Merck Group (1 mentions)
Brilliance uti agar, by Thermo Fisher Scientific (1 mentions)
Columbia agar 5 sheep blood, by bioMérieux (1 mentions)
Dulbecco s phosphate buffered saline, by Thermo Fisher Scientific (1 mentions)
Vitek 2, by bioMérieux (1 mentions)
Chapman agar, by bioMérieux (1 mentions)
Brain heart infusion broth, by BD (1 mentions)
Trypticase soy broth (tsb), by BD (1 mentions)
Api 20a system, by bioMérieux (1 mentions)
Sabouraud dextrose agar medium, by Merck Group (1 mentions)
14 protocols using sheep blood agar
1
Microbial Identification Protocols in Ingolstadt Hospital
2
Pediatric Blood Culture Protocol
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From each child, 1–3 ml of venous blood was directly collected in a paediatric blood culture bottle (BD BACTEC Peds Plus™/F, Becton Dickinson and Company, Sparks, Maryland, USA) and incubated in a BACTEC 9050 instrument (Becton Dickinson) for a total of 5 days. If flagged for bacterial growth, the cultures were subsequently Gram stained, sub-cultured on Eosin-Methylene Blue (EMB) agar, 5% Sheep Blood agar (bioMérieux, Marcy-l’Etoile, France) and chocolate + isovitalex agar, and incubated at 35–37 °C for 24 h. Isolates were identified by standard microbiological methods.
3
Culturomics Approach for Bacterial Isolation
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The standard and optimal conditions for the culturomics approach were used, based on previous work performed in our laboratory, notably for research on the human gut microbiota [14 ]. This technique started with pre-incubation: a special liquid media comprising 15 g/l brain heart infusion (Becton, Dickinson and Company, Sparks, MD 21152 USA; 38800 Le Pont-de-Claix, France), 5 g/l Bacto yeast extract (Becton, Dickinson and Company, Sparks, MD 21152 USA; 38800 Le Pont-de-Claix, France), 5 g/l proteose peptone (Oxoid Ltd, Basingstoke, Hampshire, England), 1000 ml of sterile water (Fresenius Kabi France, 5 Place de Marivel, 92310 Sèvres, France) and 5 % (v/v) sheep blood in aerobic and anaerobic conditions at 28 °C for 1 month. We inoculated them on 5 % (v/v) sheep blood agar (bioMérieux, Marcy l’Etoile, France) after performing ten serial dilutions from 1/10 to 1/10-10, allowing the growth of fastidious bacteria in order to isolate a maximum of bacterial species. This operation was carried out every 5 days from day 1 to day 25 (i.e. D1, D5, D10, D15, D20 and D25). Bacterial colonies were then isolated on 5 % (v/v) sheep blood agar after 24 h, and submitted to mass spectrometry (MALDI-TOF MS) for identification. Bacteria not identified by MALDI-TOF MS were then submitted to molecular biology for taxonomic determination by 16S sequencing.
4
Cultivation of Anaerobic and Facultative Bacteria
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The pathogenic, facultative anaerobic bacteria Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213 were obtained from the General Hospital of Santo António – Centro Hospitalar do Porto (Porto, Portugal). Both bacteria were revived from -80°C, cultured once on 5% sheep blood agar (bioMérieux, Marcy l’Etoile, France) at 37°C for 24 h and pre-grown at 37°C overnight in pre-warmed Brain Heart Infusion Broth (BHI; Merck, Darmstadt, Germany) with shaking. These pre-grown cultures were diluted immediately before initiating the growth experiments (Harris et al., 2002 ; Lin et al., 2010 (link)).
Strictly anaerobic ruminal bacteria Butyrivibrio fibrisolvens JW11, Butyrivibrio proteoclasticus P18 and Propionibacterium acnes DSMZ 1897 were from the culture collection held at the Rowett Institute of Nutrition and Health (Aberdeen, UK). The type strain of P. acnes DSMZ 1897 was originally obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany) and B. fibrisolvens JW11 and B. proteoclasticus P18 were isolated from sheep (Wallace and Brammal, 1985 (link); Wallace et al., 2006 (link)). All transfers and incubations were carried out under O2-free CO2 at 39°C in Hungate-type tubes (Hungate, 1969 ). Inoculum volumes were 5% (v/v) of a fresh culture into 10 mL of medium [liquid form of M2 medium (Hobson, 1969 )].
Strictly anaerobic ruminal bacteria Butyrivibrio fibrisolvens JW11, Butyrivibrio proteoclasticus P18 and Propionibacterium acnes DSMZ 1897 were from the culture collection held at the Rowett Institute of Nutrition and Health (Aberdeen, UK). The type strain of P. acnes DSMZ 1897 was originally obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany) and B. fibrisolvens JW11 and B. proteoclasticus P18 were isolated from sheep (Wallace and Brammal, 1985 (link); Wallace et al., 2006 (link)). All transfers and incubations were carried out under O2-free CO2 at 39°C in Hungate-type tubes (Hungate, 1969 ). Inoculum volumes were 5% (v/v) of a fresh culture into 10 mL of medium [liquid form of M2 medium (Hobson, 1969 )].
5
Bacterial Identification and Antibiotic Susceptibility
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Sample processing followed the hospital’s protocol, using specific media: sheep blood agar (bioMérieux, Marcy–l’ Étoile, France), Brilliance™ UTI Agar (Thermo Fisher Scientific, Waltham MA, USA). The strains were identified to the species level using a Vitek® 2 Compact (bioMérieux, Marcy–l’ Étoile, France) GP card and with the antibiotic susceptibility performed using Vitek® 2 Compact (bioMérieux, Marcy–l’ Étoile, France) AST P592 card.
6
Nitrite Reductase Activity Assay
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Nitrite reductase activity was monitored by nitrite consumption according to published protocols [26 (link)] with slight modifications. For anaerobic growth, meningococcal cultures on sheep blood agar (bioMérieux) were grown aerobically overnight at 37°C and 5% CO2. Meningococci that are capable of nitrite respiration grow in a characteristic halo around the nitrite disk, while meningococci that are incapable of nitrite respiration do not grow.
7
Comprehensive Microbial Culture Protocols
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All samples were cultured in eight different agar media (Additional file 8 : Table S1) as previously described [13 (link)]. In addition, for samples from the stomach, duodenum and ileum, culturing was performed on a ninth medium and for the right and left colon samples, a heat shock (20 min at 80 °C) was also performed before inoculation on Columbia agar with sheep blood (Columbia agar + 5% sheep blood, bioMérieux, Marcy l’Etoile, France). To perform direct seeding, the fresh samples were serially diluted with 0.1 M PBS (Dulbecco’s Phosphate-Buffered Saline, ThermoFisher Scientific, Paisley, UK) and immediately spread on the different culture media. All Petri dishes were incubated at 37 °C and were subcultured under the same conditions after 3, 7 and 10 days. For the samples that underwent liquid enrichment, plating and subcultures were performed on 5% sheep blood agar (bioMérieux) under anaerobic conditions on days 1, 3, 7, 10, 15, 21 and 30.
8
Bacterial Isolation and Antibiotic Susceptibility Testing
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Following the analysis of the Gram stain slides, a 10 μL sample from the ETA was inoculated onto corresponding culture media for bacterial isolation. The following media were used: sheep blood agar, chocolate agar, MacConkey agar, Chapman agar, and Sabourad dextrose agar (bioMerieux, Marcy-l’Étoile, France). The plates were incubated at 37 °C for 24 h. The bacterial growth was assessed according to the number of colony-forming units (CFU)/mL. A threshold of 105 CFU/mL was set for positive samples.
A VITEK® 2 (bioMerieux, Marcy-l’Étoile, France) antibiotic susceptibility testing system was used. A well-isolated bacterial colony was selected from the culture plate and emulsified in a sterile saline tube. The bacterial suspension was transferred onto specific cassettes following the manufacturer’s instructions, and automated susceptibility testing was performed. Antimicrobial testing was interpreted according to the Clinical and Laboratory Standards Institute (CLSI) [47 (link)].
A VITEK® 2 (bioMerieux, Marcy-l’Étoile, France) antibiotic susceptibility testing system was used. A well-isolated bacterial colony was selected from the culture plate and emulsified in a sterile saline tube. The bacterial suspension was transferred onto specific cassettes following the manufacturer’s instructions, and automated susceptibility testing was performed. Antimicrobial testing was interpreted according to the Clinical and Laboratory Standards Institute (CLSI) [47 (link)].
9
Standard Bacterial Strains for In Vitro Studies
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Standard strains of S. aureus ATCC 29213, S. epidermidis ATCC 35984, E. coli ATCC 25922, P. aeruginosa ATCC 27853, and K. pneumoniae ATCC 700603 were used as reference strains for this in vitro study. All strains were maintained at −80°C ± 2°C in brain–heart infusion broth (Difco Laboratories, MD, USA) supplemented with glycerol and checked for purity on sheep blood agar (BioMerieux, France) before use. Before experiments, the standard strains were subcultured into trypticase soy broth (TSB; Difco Laboratories, MD, USA) and incubated for 24 h at 37°C.
10
Actinomyces israelii Abscess Identification
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The Gram stain revealed moderate polymorphnuclear leukocytes and rare Gram-positive branching rods. The abscess was inoculated onto 5% sheep-blood agar, Schaedler agar, and broth (BioMérieux, France). Gram-positive bacilli were recovered after 48-hour incubation.
The colonies were very firm and notably adherent to the agar surface. They had R-type white opaque colony morphology of dome shape. A negative catalase and pigment test with no formation of indole helped us to differentiate Actinomyces from Propionibacterium species. The bacteria were identified as Actinomyces israelii (98.2% confidence level) by the API 20A system (BioMérieux, France). The bacteria were found to be susceptible to penicillin by E-test according to recommendations of CLSI. The histologic examination did not reveal any clusters of the microorganism. His informed consent was taken.
The colonies were very firm and notably adherent to the agar surface. They had R-type white opaque colony morphology of dome shape. A negative catalase and pigment test with no formation of indole helped us to differentiate Actinomyces from Propionibacterium species. The bacteria were identified as Actinomyces israelii (98.2% confidence level) by the API 20A system (BioMérieux, France). The bacteria were found to be susceptible to penicillin by E-test according to recommendations of CLSI. The histologic examination did not reveal any clusters of the microorganism. His informed consent was taken.
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