Campygen

The study was performed at the clinical microbiology laboratory of the Baruch Padeh Medical Center, Poriya, in northern Israel. From May 2015 to February 2017, a total of 66 Campylobacter isolates were collected from feces samples of patients admitted to the hospital and diagnosed as suffering from Campylobacteriosis. Forty-four of the patients were children and 22 were adults. All feces samples were routinely subcultured on Campylobacter selective agar (BD Diagnostics, Sparks, MD) and incubated at 42 °C for 48 h with CampyGen™ (Thermo Fischer Scientific, MA, USA) sachet for the generation of microaerophilic conditions.

Gastrointestinal pathogens were detected in fecal samples using a culture-based method until February 2020 and with a PCR-directed culture method using Amplidiag (Mobidiag, Ltd.). To identify Campylobacter before the introduction of Amplidiag PCR, selective Campylobacter-agar (Neogen) was used, and CampyGen (Thermo Scientific) was used to achieve a microaerophilic environment. After the introduction of Amplidiag PCR, Campylobacter was only cultured from fecal samples upon specific requests. When the primary culture was used and when a PCR-directed culture was the main method, Salmonella and Shigella were cultured when detected. The culture was based on xylose-lysine-deoxycholate (XLD) agar, Salmonella CHROMAgar (CHROMAgar), and Rappaport broth used for enrichment of Salmonella. In addition to MALDI-TOF MS, agglutination using antisera (SSI Diagnostica for Salmonella and SIFIN Antisera for Shigella, earlier from Reagensia for both) was used for Salmonella and Shigella typing.

For this study, C. jejuni strains from 45 patients having symptoms of diarrhoea were collected from 4 independent microbiology laboratories in Croatia.
A total of 45 strains, consisting of 10 strains found in primary sterile samples (9 from blood cultures and 1 from cerebrospinal fluid, samples ZGI01-ZGI10) and 35 found in stool were studied and analysed. All strains originating from primary sterile samples together with 10 strains from stool samples were collected in Zagreb (the University Hospital for Infectious Diseases “Dr. Fran Mihaljević”, samples ZG01-ZG10), another 10 strains from stool samples were received from Split (Public health Institute of Split and Dalmatia, samples ST2-ST12), 1 from Osijek (University Hospital Center Osijek, sample OS01), and 11 from Pula (Teaching Institute for Public Health County of Istria, samples PU01-PU11). Once received in the Laboratories of the Croatian Veterinary Institute, strains were streaked and sub-cultured on blood agar supplemented with 10% of defibrinated sheep blood (Columbia agar, Biomerieux, Marcy-l’Étoile, France) and incubated in microaerobic conditions (CampyGen, Thermo Scientific, Waltham, MA, USA) at 42 °C for 48 h.

A laboratory reference strain C. albicans SC5314 and six Candida clinical strains (C. albicans A14 and A69, C. tropicalis T18S and T38R, and C. parapsilosis P149 and P152) were used in the study. The six clinical strains were initially isolated from blood culture samples, retrieved from our archival laboratory collection and selected because of their strong biofilm-forming ability. All strains were identified with matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) using a Bruker Biotyper (Bruker, United States) and the manufacturer’s database (MBT Compass Library DB-7311) with a score of ≥1.7 (Vlek et al., 2014 (link)). Prior to experiment, Candida cells were first subcultured from glycerol stock culture onto Sabouraud dextrose agar (SDA) and incubated at 35°C for at least 1 day to assess the purity and viability of the culture.
Type strains L. rhamnosus GG ATCC 53103 (LGG), L. plantarum ATCC 8014 (LP8014), and L. acidophilus ATCC 4356 (LA4356) were used. Stock cultures were prepared by resuspending bacterial cells in de Man, Rogosa and Sharpe (MRS) broth with 10% glycerol and stored at −80°C until use. Before experiments, the bacterial cells were first streaked from stock culture onto MRS agar and incubated at 37°C under microaerophilic conditions (5% O₂, provided by CampyGen, Thermo Fisher Scientific, United States) for 2 days.

Bacterial strains, plasmids and primers used in this study are listed in Table 1 and Supplementary Tables S1, S2. Helicobacter pylori strains (26695 and N6) were grown on Blood Agar base no. 2 (BA) plates (Merck) supplemented with 10% (v/v) horse blood and Helicobacter Selective Supplement-Dent (ThermoFisher Scientific), or on Mueller Hinton Broth (MH) supplemented with 10% (v/v) Fetal Bovine Serum (FBS) (Lonza), at 37°C under microaerobic conditions that were provided by Anoxomat Mark II OP (MART® Microbiology B.V) or CampyGen (ThermoFisher Scientific). For the selection of H. pylori N6 hp0231::cat complemented strains, kanamycin (25 μg ml⁻¹) or/and chloramphenicol (10 μg ml⁻¹) was added to the growth media. The H. pylori N6 hp0231::cat was employed for complementation experiments by HP0231 and its mutated forms. E. coli strains were grown at 37°C on solid or liquid Luria-Bertani (LB) medium or on M63 minimal medium (Hiniker et al., 2005 (link)). When needed, media were supplemented with antibiotics at the following concentrations: 100 μg ml⁻¹ ampicillin, 30 μg ml⁻¹ kanamycin and 20 μg ml⁻¹ chloramphenicol. The E. coli strains JCB817 (dsbA::kan) and JCB818 (dsbAB::kan) (Bardwell et al., 1991 (link)), PL263 (mdoGdsbC::kan) (Leverrier et al., 2011 (link)) were employed for complementation experiments by HP0231 and its mutated forms.

Bacterial strains and plasmids are listed in Tables S1. Bacterial cultures were routinely maintained in Luria-Bertani (LB) liquid medium or on LB agar plates. M9 medium with glucose (22 mM) was used as minimal medium. Microaerobic and anaerobic environments were produced using the CampyGen (Thermo Scientific) and the GasPak (BD Diagnostics) sachets gas generating systems, that were place in sealed containers without shaking. Media were supplemented with the appropriate antibiotics at the following concentrations: tetracycline 20 μg/mL, kanamycin 50 μg/mL, and ampicillin 100 μg/mL, when appropriate.

Growth at various temperatures (28 °C, 37 °C, 42 °C, 45 °C) in different atmospheres (aerobic, microaerophilic using CampyGen from Thermo Scientific and anaerobic using AnaeroGenTM from bioMérieux) was tested by culture on Columbia agar (bioMérieux) after 48 h of incubation. The salinity acceptance limit of SN6^T strain was investigated by culture on a home-made culture medium consisting of a Columbia agar culture medium (Sigma-Aldrich, Saint-Quentin Fallavier, France) modified by adding (per liter) 5 g MgCl2 6H2O, 5 g MgSO4 7H2O, 2 g KCl, 1 g CaCl2 2H2O; 0.5 g NaBr, 0.5 g NaHCO3, and 2 g glucose with various NaCl concentrations 0, 5, 10, 25, 50, and 75 g/L. The pH range (6; 6.5; 7; 8.5) for growth was also determined and pH was adjusted by addition of HCl or NaOH.