The molecular epidemiology of the isolates was determined by repetitive sequence-based PCR (DiversiLabTM System; bioMérieux).17 (link) Clonal relatedness was analysed with the DiversiLab software using the Pearson correlation statistical method. Isolates showing ≥98% similarity in their rep-PCR pattern were considered identical. Using the modified Kullback–Leibler statistical method, isolates were assigned to international clones (IC) using our in-house database and isolates that clustered at >95% were considered related to the IC.17 (link)
Diversilab system
Manufactured by bioMérieux
Sourced in France
The DiversiLab system is a molecular typing platform designed for microbial strain comparison and identification. It utilizes repetitive-sequence-based PCR technology to generate DNA fingerprints that can be used to compare and analyze microbial isolates.
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13 protocols using diversilab system
1
Molecular Epidemiology of Bacterial Isolates
2
Genetic relatedness analysis of pAmpC-E
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Genetic relatedness between pAmpC-E that persisted at one or two month was assessed by repetitive sequence-based PCR (rep-PCR) using the semi-automated DiversilabTM system (BioMérieux, Marcy-l’Étoile, France). The amplified products were separated by electrophoresis on microfluidics chips and analyzed with the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). The peak and band data were analyzed by DiversiLabTM software (bioMérieux). The cut-off value was 95% for determining genetic similarity.
3
Identifying Acinetobacter baumannii Colony Diversity
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To determine how many colonies from a patient culture plate should be analyzed,
we hypothesized that there are different colony morphotypes of
A. baumannii on CHROMagar and each colony
morphotype represented a different genetic type. To test this hypothesis, in a
preliminary pilot study, we tested colonies from 12 patients. The isolates were
typed by repetitive sequence-based PCR (DiversiLabTM System;
bioMérieux). Clonal relatedness was analyzed with the DiversiLab software using
the Pearson correlation statistical method. Relatedness was defined as: ≥98%
similarity as identical, ≥ 95% and <98% similarities as related, and <95%
similarity as unrelated.
we hypothesized that there are different colony morphotypes of
A. baumannii on CHROMagar and each colony
morphotype represented a different genetic type. To test this hypothesis, in a
preliminary pilot study, we tested colonies from 12 patients. The isolates were
typed by repetitive sequence-based PCR (DiversiLabTM System;
bioMérieux). Clonal relatedness was analyzed with the DiversiLab software using
the Pearson correlation statistical method. Relatedness was defined as: ≥98%
similarity as identical, ≥ 95% and <98% similarities as related, and <95%
similarity as unrelated.
4
Molecular Characterization of MRSA Isolates
5
Bacterial Identification and Antimicrobial Susceptibility
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Semi-quantitative counts of bacterial growth were performed using the adequate plate and the following formula: colony counts on Petri dishes × inverse of dilution factor × 10 = number of colony-forming units (CFU)/mL of swab initial suspension. Data were expressed as log10 CFU/mL.
After counts, each different phenotype was re-isolated on Brain Heart Infusion (BHI) or Mannitol salt agar (S. aureus) and then submitted to catalase test and Gram stain. Identification was performed using specific cards of the VITEK 2® system for Gram-positive and Gram-negative bacteria (bioMérieux, Marcy-l’Etoile, France). The repetitive extragenic palindromic sequence–based polymerase chain reaction (rep-PCR) using DiversiLab system (bioMérieux) was used according to the manufacturer’s recommendations to compare strain similarity.
Antimicrobial susceptibility test was performed using Bauer–Kirby method with the following antimicrobials: vancomycin (Enterococcus spp.); imipenem (P. aeruginosa, K. pneumoniae, and A. baumannii); third generation of cephalosporin, ceftazidime, cefotaxime, ceftriaxone, and aztreonam (K. pneumoniae); and cefoxitin (S. aureus). Agar dilution with vancomycin was used for S. aureus according to Clinical and Laboratory Standards Institute (CLSI).6
After counts, each different phenotype was re-isolated on Brain Heart Infusion (BHI) or Mannitol salt agar (S. aureus) and then submitted to catalase test and Gram stain. Identification was performed using specific cards of the VITEK 2® system for Gram-positive and Gram-negative bacteria (bioMérieux, Marcy-l’Etoile, France). The repetitive extragenic palindromic sequence–based polymerase chain reaction (rep-PCR) using DiversiLab system (bioMérieux) was used according to the manufacturer’s recommendations to compare strain similarity.
Antimicrobial susceptibility test was performed using Bauer–Kirby method with the following antimicrobials: vancomycin (Enterococcus spp.); imipenem (P. aeruginosa, K. pneumoniae, and A. baumannii); third generation of cephalosporin, ceftazidime, cefotaxime, ceftriaxone, and aztreonam (K. pneumoniae); and cefoxitin (S. aureus). Agar dilution with vancomycin was used for S. aureus according to Clinical and Laboratory Standards Institute (CLSI).6
6
Screening and Genotyping of ESBL-E
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Screening swabs (soaked with Amies transport medium) were taken from the rectum and cultivated on ChromID ESBL agar (bioMérieux, Nürtingen, Germany) selecting for ESBL-E. Species identification and antibiotic susceptibility testing of bacteria grown on ChromID ESBL agar was performed by Vitek 2 GN ID and AST N223 card (bioMérieux), respectively. Isolates were included in the study, if they tested non-susceptible to cefotaxime, ceftriaxone or ceftazidime using EUCAST breakpoints [24 ].
The combination disc test following EUCAST guidelines using cefotaxime, ceftazidime and cefepime with and without clavulanate (Mast Diagnostica, Reinfeld, Germany) was performed to confirm ESBL production [24 ].
Genotyping of 3GCREB isolates was conducted by repetitive-sequence-based PCR and subsequent microfluidics electrophoresis using the DiversiLab system (bioMérieux).
The combination disc test following EUCAST guidelines using cefotaxime, ceftazidime and cefepime with and without clavulanate (Mast Diagnostica, Reinfeld, Germany) was performed to confirm ESBL production [24 ].
Genotyping of 3GCREB isolates was conducted by repetitive-sequence-based PCR and subsequent microfluidics electrophoresis using the DiversiLab system (bioMérieux).
7
Molecular Profiling of Antibiotic-Resistant Staphylococcus
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Genes conferring resistance to MLS [erm(A), erm(B), erm(C), and msr(A)], aminoglycosides [aph(3′)-IIIa, ant(4′)-Ia, and aac(6′)-aph(2″)] and tetracyclines [tet(M) and tet(K)] were screened by PCR, as previously described [27 (link)–29 (link)]. Mechanisms of fluoroquinolone resistance were studied by sequencing QRDRs of gyrA and parC genes [30 (link)] and by determining MICs of ciprofloxacin with or without reserpin (10 μg/ml). Both pvl and tst genes coding for Panton-Valentine leukocidin (PVL) and toxic shock staphylococcal toxin (TSST-1), respectively, were screened by PCR as previously described [31 (link)–33 (link)].
For molecular typing, four different techniques were used for all the strains. The MLST was performed as previously described [34 (link)] using the MLST database (http://saureus.mlst.net/ ). The spa typing was carried out as previously described [35 (link)] using the Ridom StaphType software (Ridom GmbH, Würzburg, Germany). The typing of staphylococcal cassette chromosome mec element (SCCmec) was performed according to mec and ccr complexes previously defined [36 (link)]. Genetic relatedness was determined by rep-PCR using the semi-automated Diversilab system (bioMérieux, Marcy l’étoile, France) [14 (link), 37 (link)].
For molecular typing, four different techniques were used for all the strains. The MLST was performed as previously described [34 (link)] using the MLST database (
8
Molecular Profiling of Staphylococcal Infections
9
Epidemiological Typing of A. baumannii
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Epidemiological typing of A. baumannii strains from ICUs was performed by repetitive-PCR typing using DiversiLab system (bioMerieux, France) as previously described (Chmielarczyk et al. 2016 (link)). Isolates that clustered ≥91.3% were considered related. Results of A.baumannii typing were already published.
10
Genetic Relatedness Analysis of Acinetobacter
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Genetic relatedness of the isolates was assessed using semi-automated rep-PCR on the DiversiLab® System (bioMérieux) after following the manufacturer’s guidelines. Data were analyzed and interpreted using internet-based DiversiLab software (v. 3.4) with the Kullback-Leibler correlation coefficient. Strains presenting a similarity of ≥95% were considered clonally related [13 (link)]. Multilocus sequence typing (MLST) was performed on A. baumannii isolates that were not susceptible to carbapenems following protocols previously described by Bartual et al. 2005 [21 (link)] and Diancourt et al. 2010 [22 (link)] available at the PubMLST website (http://pubmlst.org/acinetobacter , accessed on 30 December 2020).
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