Microbial Monitoring in Controlled Environment

The following HAI-related microorganisms were monitored: Staphylococcus spp. and Staphylococcus aureus, Enterobacteriaceae, Acinetobacter, Pseudomonas spp., Clostridium difficile, Candida spp. and Aspergillus spp. A total of 360 microbiological samples were collected in duplicate (720 total samples). The following growth media were used: the Tryptic Soy Agar with Lecithin, Tween and Histidine (Merck Millipore, Darmstadt, Germany) general growth medium was used for total bacterial count; Baird Parker Agar (Merck Millipore, Darmstadt, Germany), moderately selective medium for coagulase-positive staphylococci; MacConkey Agar (Merck Millipore, Darmstadt, Germany), selective for Enterobacteriaceae; Chromatic^TM Agar (Liofilchem®—Italy) for Acinetobacter species detection; Cetrimide Agar (Cetrimide agar base, BD Diagnostic Systems), selective for Pseudomonas spp.; Clostridium difficile Agar (Ref. 31044 Clostridium selective agar Lickson—Italy), selective for Clostridium difficile; Sabouraud Dextrose Contact Agar with chloramphenicol (Merck Millipore, Darmstadt, Germany), selective for Mycetes and Candida albicans. Incubation was performed aerobically at 37°C (48–72 hours) for Baird Parker, MacConkey, Cetrimide, Chromatic^TM Agar and anaerobically Clostridium difficile Agar by using anaerobic jars (GasPak™, Thermo Fisher Scientific Inc.) with AnaerobicGen^TM System (Thermo Fisher Scientific Inc.) at 37°C for 72 hours. Colony Forming Units (CFU) on all agar plates were manually counted after their respective incubation period. Identification of isolates was assessed by Staph System (Liofilchem—Italy) for Staphylococcus aureus, Entero Pluri Test (Liofilchem—Italy) for Escherichia coli, Oxi/Ferm Pluri Test (Liofilchem—Italy) for Pseudomonas aeruginosa and API 20 C Aux (bioMérieux, Inc) for Candida albicans. After incubation under anaerobic checking and detection of the Clostridium difficile was assessed by Latex Agglutination test (Liofilchem—Italy).
Bacillus isolates were obtained from Tryptic Soy Agar plates. At least 20 isolates per each sampling time were inoculated in 5 ml LB and expanded for 24 hours at 37°C for subsequent analyses.