MALDI-TOF mass spectrometry analysis and molecular typing by pulsed-field gel electrophoresis of environmental Vibrio isolates
Rkia Eddabra1,2, Jean-Michel Scheftel2, Gilles Prévost2, Olivier Meunier3, Rachida Mimouni1*
1Laboratoire Biotechnologies et Valorisation des Ressources Naturelles (LBVRN), Faculté des Sciences, Université Ibn Zohr, B.P. 8106, Agadir, Maroc.
2UPRES EA-3432, Laboratoire de Bactériologie de la Faculté de Médecine, Université Louis Pasteur, Hôpitaux Universitaires de Strasbourg.
3Laboratoire d'hygiène hospitalière, Hôpitaux Universitaires de Strasbourg, 67091 Strasbourg, France
*Corresponding autor E-mails: eddabra@gmail.com, r_mimouni@yahoo.fr
Abstract
Pulsed field gel electrophoresis (PFGE) is widely used as an epidemiological tool for the typing and identification of bacterial strains. In contrast, Matrix-Assisted Laser Desorption Ionisation/Time-of-Flight Mass Spectrometry (MALDI-TOF MS) offers a new and promising approach to rapid identification and differentiation of bacterial strains. 30 Vibrio strains were isolated from two wastewater treatment plants from Agadir (14 V. cholerae non-O1, 10 V. alginolyticus, 5 V. fluvialis and one strain of V.
metschnikovii) identified by Vitek 2 system (bioMérieux). 50% of all isolates are susceptible to all antibiotics tested. Differentiation of Vibrio strains was done using PFGE of NotI-digested genomic DNA. In addition MALDI-TOF-MS was performed.
Out of the 30 strains of Vibrio examined in this study, 6 isolates could not be typed by PFGE and consistently appeared as a smear on the gel. In general, high genetic biodiversity among the Vibrio strains was found regardless to the isolation source. The results of MALDI TOF analysis show that the spectral profile, consisting of about 20 to 25 prominent peptide peaks masses ranging between 2 and 12 kDa. MALDI-TOF mass spectrometry could cluster the isolates from each species into the same pattern, whereas PFGE had lower discriminatory ability between species.
Key words: Antibiotic resistance, MALDI TOF, PFGE, Vibrio, Wastewater.
Introduction
The Vibrionaceae are gram- negative Gammaproteobacteria that occur in temperate to tropical, coastal, and estuarine marine systems (Thamposon et al. 2004). Vibrio spp. is autochthonous to a diverse range of ecological niches, including estuarine, coastal waters and sediments, the water column, and in association with organisms either as symbionts (Ruby et al., 2005) or pathogens (Makino et al.
2003).
The diversity of Vibrio species has been examined using multiple molecular techniques including multilocus sequence analysis (MLSA), pulsed-field gel electrophoresis (PFGE), and group specific PCR (GS-PCR) analysis. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), have been established for monitoring biomarkers in bacterial extracts.
MALDI-TOF MS, this method is based on the detection of mainly ribosomal protein fractions of bacteria, which are
the most abundant and conserved (Lay, 2001).
New developments and enhancements of this technology were done in order to characterize a wide spectrum of microbial cells. MALDI- TOF Mass Spectrometry has been shown high potentialities to discriminate taxa very close related (Dieckmann et al., 2008; Sauer and Kliem, 2010).
In this work, we investigated the ability of MALDI-TOF-MS to discriminate between closely related environmental strains of Vibrio and to characterize them by pulsed field gel electrophoresis (PFGE).
Materials and methods
Wastewater samples were collected from two municipal wastewater treatment plants (Ben Sergao and Drarga) located in the Agadir city (Morocco). Thereafter, Vibrio strains isolated in wastewater samples on TCBS medium (Difco), are identified by VITEK 2 system (bioMérieux). Furthermore, all isolates of Vibrio were examined for their susceptibility to 20 antimicrobials (BIO-RAD), Vitek 2 GN® card was used with Vitek 2 system, version VT2- XL 135P. In addition strains of Vibrio identified as V. cholerae were confirmed serologically by the slide agglutination test using specific polyvalent antiserum to V. cholerae O1 (Bio-Rad, Marnes-la-Coquette-France).
Then Bacteria were cultured in CAYE medium, and the titers of cholera toxin (CT) were determined by reversed passive latex agglutination (RPLA) according to manufacturer’s recommendations (Oxoid toxin, detection kit VET-RPLA).
Antimicrobial susceptibility testing was done using standard
methods (disc diffusion method) using Mueller–Hinton agar, and interpreted according to the antibiogram guidelines of the French Committee of Microbiology (Soussy, 2005).
Antimicrobials used for testing were ampicillin (AMP) 10 µg; cephalothin (CEF) 30 µg; ticarcillin (TIC) 75 µg;
cefotaxime (CTX) 30 µg; amoxicillin (AMX) 30 µg; cefoxitin (FOX) 30 µg;
amoxicillin+clavularic acid (AMC) 20/10 µg; trimethoprim–
sulfamethoxazole (SXT) 1,25/23,75·µg;
gentamicin (GEN) 15 µg; nalidixic acid (NAL) 30 µg; ciprofloxacin (CIP) 5 µg;and ofloxacin (OFX) 5 µg.
PFGE analysis was performed with Genepath® Group 3 Reagent Kit (Bio-Rad). For this, the genomic DNA embedded within agarose plugs was digested with NotI restriction endonuclease. After, Digitized gel images were analyzed by means of the Fingerprinting II software (Bio-Rad).
PFGE relationships were defined using Tenover’s criteria. Percent similarity of macrorestriction patterns was estimated with the dice coefficient, and the dendrogram was clustered by the unweighted pair group method.
MALDI-TOF-MS analysis
Sinapinic acid matrix solution was prepared at a concentration of 10 mg/ml in acetonitrile (70% v:v) and 2.5%, Trifluoroacetic acid (TFA) (30% v:v).
Each Vibrio isolates was removed using a sterile wooden toothpick. The cells were spotted directly onto a 384 well stainless steel target plate and immediately overlaid with 1.0 µl of the matrix solution. Following air drying, the samples were analyzed using a Bruker Ultraflex II MALDI-TOF-MS (Bruker Daltonics). The instrument was operated in positive linear, delayed extraction mode with an acceleration voltage of 20 kV. Prior to data
collection, the mass spectrometer was externally calibrated and each mass spectrum was acquired by accumulating 200 laser shots. Mass range of 2,000 – 20,000 m/z was used for analyzis.
MALDI-TOF-MS data analysis
Automated spectrum acquisition was performed using the Auto ExecuteTM software (Bruker Daltonics) with fuzzy control of laser intensity.
Spectra analysis generated data that included both peak position and intensity. Mass spectra were analyzed with Flex Analysis software 2.4 (Bruker Daltonics). Further bacterial data analyses were performed by BioTyperTM 2 software developed by Bruker Daltonics.
For the comparison of Vibrio strains in general, 75 peaks were picked automatically. PCA (principal compound analysis), was used to calculate their similarity. The hierarchical clustering uses a dendrogram based algorithm to form tree-like structures from the distance of Vibrio strains (the scores of PCA) and links them together by a linkage algorithm.
Results
Identification of Vibrio isolates
A total of 30 isolates of Vibrio strains were isolated from wastewater (raw, decanted and treated ) of two
wastewater treatment plants in Agadir, which correspond to 14 V. cholerae non O1, 10 V. alginolyticus, 5 V. fluvialis and one strain of V. metschnikovii. All V. cholerae strains belonged to non-O1 serogroup and did not produced the cholera toxin (CT).
Antimicrobial susceptibility
Results of antimicrobial susceptibility testing for isolates are summarized in Table 1. 85% of V.
cholerae and 40% of V. alginolyticus are susceptible to all antibiotics tested;
all isolates of V. fluvialis are amoxicillin and cefoxitin resistant since 60% of V.
alginolyticus and V. metschnikovii are amoxicillin and ticarcillin resistant.
Genotyping by PFGE
Analysis of genomic DNA by pulsed-field gel electrophoresis (PFGE) has been used to characterize clonal diversity and relationships among Vibrio isolates. This method allows the distinction of large fragment, reflecting genome variation among epidemic strains. The NotI restriction enzyme transforms the chromosomal genome into 11 to 21 fragments which ranged from 48.5 to 533 kb. In this study 5 of 30 strains (3 V. cholerae, 2 V.
alginolyticus) could not be typed by PFGE and appear as a smear, even when thiourea was added to running buffer.
Table 1. Antimicrobial resistance phenotypes for Vibrio sp. strains isolated in two wastewater treatment plants (WWTP) at Agadir.
AMX: Amoxicillin; FOX:
Cefoxitin; TIC: Ticarcillin
Vibrio strains Antibiotic resistance profil
Number of strains
Total
V. cholerae Susceptible to all antibiotics AMX, TIC
12
2 14
V. alginolyticus Susceptible to all antibiotics AMX, TIC
4
6 10
V. fluvialis AMX, FOX 5 5
V. metschnikovii AMX, TIC 1 1
Figure 1 shows a dendrogram constructed using the UPGMA method, based on PFGE band patterns obtained
using NotI. Vibrio Isolates were classified into XV clusters.
Dice (Tol 0.5%-0.5%) (H>0.0% S>0.0%) [0.0%-100.0%]
100
80
6040
20 50.00
100.00
150.00
200.00
250.00
300.00
350.00
400.00
500.00
2000
5.00E3
Kb
V. cholerae V. alginolyticus V. cholerae V. fluvialis V. cholerae V. cholerae V. fluvialis
V. Alginolyticus V. cholerae V. cholerae V. alginolyticus V. cholerae V. cholerae V. cholerae V. alginolyticus V. fluvialis V. cholerae V. metschnikovii V. alginolyticus V. fluvialis V. fluvialis V alginolyticus
V. alginolyticus lambda ladder lambda ladder V. cholerae V. alginolyticus
I II III IV V
VI VII
VIII IX X
XI XII
XIII XIV XV
Figure 1: Dendrogram based on the cluster analysis of Vibrio strains isolated in wastewater treatment plant of Agadir, NotI macrorestriction patterns resolved by PFGE
MALDI MS profiling was used for taxonomic differentiation of 30 Vibrio isolates. The MALDI BioTyper analysis software incorporates functionalities for the processing of mass spectra, as well as for identification and classification of the respective microorganisms (Bruker Daltonics Application Note MT-80, 2005).
The spectral patterns obtained from whole cell analysis gives characteristic fingerprints, which are thought to be from the desorbed components of the cell envelope, a typical MALDI-TOF spectrum of Vibrio sp. contained about 150 ion peaks between 2000 and 20000 Da, with the highest intensity peaks found between 2000 and 11000 Da. Figure 2 shows an example of MALDI-TOF spectra in range 2–4 kDa, in which each strain of Vibrio is defined by its unique mass distribution corresponding to a fingerprint spectrum characteristic of the culture conditions used. The spectra
show many interspecies similarities (some peaks were common in spectra of all isolated Vibrio strains) i.e., peak at m/z: 2590, 3100, 3500, as well as species-specific biomarkers i.e., peak at m/z: 2400, 2800, 3800.
Using the MALDI BioTyper, PCA can be performed using multiple mass spectra obtained for each species.
Based on the calculated principle components, the four different species (V. cholerae, V. alginolyticus, V.
fluvialis and V. metschnikovii) of Vibrio isolates can unambiguously be separated by hierarchical clustering (Figure 3)
Discussion
The role of antibiotics in the management of human infections caused by Vibrio spp. has not yet been defined, although antimicrobial resistance could be an important
2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 1 2
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File no.
Multi-spectrum view
M/Z [Da]
Figure 2. Examples of MALDI-TOF mass spectra of intact cells of Vibrio isolated in wastewater treatment plants. Main masses between 2.000 and 4000 Da are indicated.
Figure 3. PCA score plot for individual species of the genus Vibrio.
problem for therapy directed against these organisms (Arai et al., 1985).
Multi-drug resistant of environmental Vibrio species have been frequently reported from many part of the world (Ibara and Alvarado, 2006). It has been observed that antibiotic
susceptibility of Vibrio spp. is dynamic and varies with the environment (Ottaviani et al., 2001; Jun et al., 2003).
This study revealed a high prevalence of antibiotic resistances in the Vibrio isolates. The resistance patterns detected ranged between two and three
drugs. The results of this study confirm the widespread diffusion of antibacterial resistances in Vibrio isolates from wastewater treatment (Mezrioui and Oufdou, 1996). It could be observed that a high percentage of the isolates tested (46, 66%) was resistant to AMX, confirming that this is the most common resistance (Akinbowale et al., 2006).
Pulsed-field gel electrophoresis has frequently been used for detailed molecular typing of many species of bacteria (Tenover et al., 1995). The results of PFGE showed that the Vibrio strains had different fragment patterns (Figure 1). On the other hand and a major concern for this study was the presence of Vibrio strains untypeable by PFGE. Strains that are untypeable may probably be a result of methylation of genomic DNA (Xydas et al., 1996), or its degradation during the process (Wong et al., 2004). Several works suggested that environmental strains are quite heterogeneous, making the technique less useful for determining relationships between strains isolated from different sources (Wong et al., 2004). Our study confirms again the high degree of diversity among PFGE patterns of Vibrio isolated in two wastewater treatment plants.
Currently MALDI-TOF mass spectrometry has been applied successfully to a wide variety of microbial applications including the analysis of bacterial RNA and DNA, the detection of recombinant proteins, the characterization of unknown proteins, bacterial proteomics, the detection of virulence markers, and the very rapid characterization of bacteria at the genus, species, and strain level (Lay, 2001).
MALDI-TOF MS of intact microorganisms has been shown to produce characteristic mass spectral fingerprints of moieties desorbed from
the cell surface (Claydon et al., 1996;
Holland et al., 1996). In the dendrogram for MALDI-TOF-MS, results are different from those obtained by PFGE.
This result can be explained by the differences in target substances analyzed by the two methods. PFGE analyzes the chromosomal DNA of bacteria for variations in the locations of specific restriction enzyme cleavage sites, whereas MALDI-TOF-MS analyzes abundant constructed molecules such as ribosomal proteins and peptidoglycans in bacteria.
Our results suggest that whole- cell MALDI-TOF MS-based proteometric characterization represents a powerful tool for rapid and accurate classification of Vibrio spp. and related species, whereas PFGE analyses take several days to complete.
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