Matrix assisted laser desorption ionization time of flight mass spectrometry

S. algae strains ACCC, YHL, and CHL were obtained from various clinical sources (Table 1). Glycerol stock of stored isolates was grown in trypticase soy agar with 5% sheep blood (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) at 30°C for 24 hours. Single colonies were inoculated in tryptic soy broth (Becton, Dickinson and Company, Franklin Lakes, NJ). The isolates were preliminarily identified using 16S rRNA gene sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (bioMérieux, Marcy l'Etoile, France). A part of 16S rRNA gene was amplified using the primers of B27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and U1492R (5′-GGTTACCTTGTTACGACTT-3′) [9 (link), 16 (link)]. The nucleotide sequences were aligned, and BLAST search was performed against the GenBank database of the National Center for Biotechnology Information (NCBI) [17 (link)].

From January 2016 to December 2018, 186 non-duplicate clinical isolates were collected from 32 hospitals of 22 provinces or cities across China, and 29 reference strains were purchased from the American Type Culture Collection¹. These clinical isolates were resistant to at least one of the carbapenem antibiotics (ertapenem, meropenem, or imipenem), including 124K. pneumoniae, 26 Escherichia coli, 23 Enterobacter cloacae, 5 Klebsiella oxytoca, 2 Citrobacter freundi, 2 Enterobacter aerogenes, 2 Serratia marcescens, 1 Morganella morganii, and 1 Providencia rettgeri. Twenty-nine reference strains (including 17 K. pneumoniae, 8 E. coli, 1 E. cloacae, 1 K. oxytoca, 1 P. rettgeri, and 1 Enterobacter hormaechei) with or without carbapenemase were involved in this study (Table 1). All tested isolates were identified to the species level using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (bioMérieux, France).

A total of three Aeromonas isolates were included in this study. The first (A-1) was from a patient's blood culture (Patient A), while the other two were isolated 2 weeks apart from the intra-abdominal abscess (B-1) and T-tube fluid (B-2) from Patient B. These three isolates were identified as Aeromonas hydrophila from the original culture plates using Matrix Assisted Laser Desorption Ionization-Time of Flight mass spectrometry (Biomerieux). The antimicrobial susceptibility testing was performed using Broth Microdilution (BMD) following guidelines set forth by the Clinical and Laboratory Standards Institute (CLSI) (CLSI, 2016 ). The Modified Carbapenem Inactivation Method (mCIM) and the EDTA-Carbapenem Inactivation Method (eCIM) were performed following the CLSI of the M100 guidelines (29th edition) (Clinical Laboratory Standards Institute, 2019 ). Modified Hodge Test (MHT) was also performed (Amjad et al., 2011 (link)). A positive control (MHT positive Klebsiella pneumoniae ATCC1705) and a negative control (MHT negative Klebsiella pneumoniae ATCC1706) were included in all the three phenotypic carbapenemase tests.

Samples were processed, and E. coli was isolated as described previously [29 (link)] using eosin methylene blue agar (BD, Sparks, MD, USA) and MacConkey agar plates (BD). Species identification was performed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (bioMérieux, Marcy l'Étoile, France).
Antimicrobial susceptibility was assessed by determining the minimum inhibitory concentrations (MICs) for 16 antimicrobial agents using the broth microdilution method with a commercially available Sensititre® panel KRVP4F (TREK Diagnostic Systems, West Sussex, UK) according to the manufacturer's instructions. The following antibiotics were tested: ampicillin, amoxicillin/clavulanic acid, cefoxitin, ceftiofur, ceftazidime, cefepime, chloramphenicol, ciprofloxacin, colistin, gentamicin, meropenem, nalidixic acid, streptomycin, sulfisoxazole, tetracycline, and trimethoprim/sulfamethoxazole. The reference strain E. coli ATCC 25922 was used as quality control when determining MICs. The MIC was interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2017). When CLSI breakpoints were not available, the MIC was interpreted according to the Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP, 2014). Multidrug resistance was defined as resistance to ≥3 antibiotic subclasses.

Rectal swabs were used for collecting the fecal material from the pets and were immediately placed in the transport medium Cary-Blair Medium (Oxoid). Within 24 h, the swabs were soaked in 1 mL of sterile saline, vortexed, and 0.1 mL was inoculated on plates of MacConkey Agar (Oxoid) containing 1 mg/L imipenem (Sigma-Aldrich). The plates were incubated aerobically at 37°C ± 1 for 48 h.
All the different morphotypes of colonies grown in the same plate were subcultivated to obtain pure culture. Isolates were identified firstly by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (bioMérieux) and successively confirmed by partial sequencing of the 16S rRNA gene (Brosius et al., 1978 (link)).

A total of 2547 S. aureus isolates (382 cattle, 1077 pig, and 1088 chicken carcass isolates) were obtained from 16 laboratories/centers participating in the Korean Veterinary Antimicrobial Resistance Monitoring System. Sample collection and isolation of S. aureus were performed as described previously [19 (link)]. Briefly, the back and chest of cattle and pig carcasses were swabbed with sterile gauze pads wetted with buffered peptone water (BPW) (Becton Dickinson, Sparks, MD, USA), while the whole carcasses of chickens were rinsed in Phosphate Buffered Water (PBW). Homogenized samples were inoculated into tryptic soy broth (Becton Dickinson) containing 6.5% sodium chloride and incubated at 37 ℃ for 16 h. Following incubation, one or two loops from each enrichment broth were streaked onto mannitol salt agar (Difco, Detroit, MI, USA). Suspected colonies were then identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Biomerieux, Marcy L’Etoile, France). S. aureus and MRSA isolates were further confirmed by a multiplex polymerase chain reaction (PCR) assay specific for the 16S rRNA, clfA, and mecA genes [40 (link)].

Seventy-one non-redundant multidrug-resistant CRKP isolates were obtained from patients attending the Microbiology Section of the King Abdulaziz Medical City, Riyadh, Saudi Arabia, between January 2011 and December 2012. The isolates were identified to the species level using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (bioMérieux, Marcy-l’Étoile, France). The frozen isolates were sub-cultured on blood agar at 37 °C overnight. Single colonies were picked up and grown in liquid tryptic soy broth(TSB) medium 37 °C in a shaking incubator at 250 rpm overnight. Minimum inhibitory concentrations (MICs) were determined using a Micro VITEK® 2 microbial identification instrument (bioMérieux). The MIC breakpoints for carbapenem were defined according to the modified 2010 Clinical and Laboratory Standards Institute guidelines [7 ]. Isolates found to have elevated MICs for carbapenem by Micro VITEK® 2 analysis were confirmed by the manual ETEST® (bioMérieux) to have reduced susceptibility to either imipenem or/and meropenem.