Enterococcus species are one of the leading causes of nosocomial infections, which are difficult to treat specially with the rise of its Vancomycin resistant. Studies of Enterococcus isolates are essential for epidemiological investigation. Typing Enterococci is often based on the traditional phenotypic as well as genotypic methods. In this study Fourier-transform infrared (FTIR) spectroscopy is used as a novel phenotypic approach to the typing of Enterococci. FTIR spectroscopy results compared to antibiotic susceptibility testing and PCR amplification of Vancomycin gene results; the analysis showed that, 6 isolates were positive for Van gene (4 of VanA, 1 of VanB and 1 VanA plus VanB). Three of VanA and VanA plus VanB were resistant to all antibiotic tested (Ampicillin, Teicoplamin and Vancomycin) and VanB was found to be sensitive. FTIR spectroscopy (first derivatives) divided the isolates into 8 groups. 3 groups of VanA (4 isolates), one of VanB (one isolate), one of VanA plus VanB (one isolate) and the other 13 Enterococcus isolates were divided into 3 clusters. The study demonstrated that FTIR spectroscopy has good discriminative capacity and high reproducibility as compared to other techniques.
Enterococci is an opportunistic human pathogens, the two most important species of this group are E. faecalis and E. faecium, which are considered as the leading causes of nosocomial infections. It also causes: endocarditis, septicemia, intra-abdominal, pelvic and urinary tract infections [
For most clinical microbiological laboratories, the primary method of identifying Enterococcus strains relies on the traditional phenotypic characterization. However, it is a challenging procedure that can take several days to accomplish because of the phenotypic and biochemical similarities between many Enterococci [
An extensive approach for investigation of bacterial cells of different bacterial species and strains has been undertaken by Neumann and his co-worker [
Attempts to apply Infrared (IR) Spectroscopy technology to biology began as early as the 1910s, when the use of IR spectroscopy for the analysis of biological samples was first suggested. By the late 1940s, the technique was being successfully explored for the study of biological materials and in fact, IR spectroscopy has become an accepted tool for the characterization of biomolecules [
FT-IR spectra of intact microbial cells are highly specific fingerprint-like signature. It is a valuable tool for the identification of biological samples of interest [
In the present work, the Fourier Transform Infrared spectroscopy [FTIRS] was used to type Enterococcus sp. that was isolated from a hospital in an attempt to develop a standardized procedure for the differentiation and characterization of Enterococci.
A total of 19 clinical isolates of Enterococcus sp. were collected from different body sites of various hospital wards. A standard strain of e. faecalis (ATCC-51299) was also inculded. The Enterococci isolates were stocked in proper media and stored at −70˚C.
Antibiotic susceptibilities were determined by the disk diffusion method on Mueller-Hinton medium (Difco Laboratories), according to the methods outlined by the Clinical and Laboratory Standards Institute, formerly the National Committee for Clinical Laboratory Standard [
DNA was extracted using rapid lysis method [
Multiplex polymerase chain reaction or Multiplex pcr was carried out with 25 µL reaction volume, each reaction contained 2.5 µL of 10X buffer, 0.5 µL of dNTP (Invitrogen, United Kingdom), 0.2 µL of VanA primer 5'-CATG AATAGAATAAAAGTTGCAATA-3', 5'-CCCCTTTAACGCTAATACGATCAA-3', and 0.1 µL of VanB primer, 5'-GTGACAAACCGGAGGCGAGGA-3', 5'-CCGCCATCCTCCTGCAAAAAA-3', 0.25 µL Taq DNA po- lymerase (Invitrogen; United Kingdom) and 3.0 µL. Thermocycling DNA was performed in PTC-150 DNA engine with an initial 5 min. denaturation at 95˚C, followed by 30 cycles of denaturation at 95˚C, for 30 seconds, annealing at 55˚C, for 1 min. and extension at 72˚C, for 1 min, followed by a final extension step at 72˚C, for 5 min.
The Enterococcus sp. was grown under standardized conditions on blood agar plates. A single isolated colony was carefully removed from previously prepared culture using a sterile 1 µL disposable loop and suspended in 200 µL sterile physiological saline solution (0.85% NaCl). Then 120 µL of the suspension was evenly spread over the entire surface of a silicon window 32 mm dia, and allowed to dry for 30 min at 39˚C, leaving a transparent film suitable for FTIR absorbance transmission measurements. It is reported that first derivatives of infrared spectra were recorded in fingerprint region 900 - 700 cm−1 [
Disc diffusion test was carried out for the three antibiotics and results are summarized in
The FTIR spectra of Enterococci samples were obtained in the range 4000 - 500 cm−1 which includes the known fingerprint region i.e., 900 - 700 cm−1. The results were repeated for each sample over different times and the results were highly reproducible over the indicated time period as shown in
Another absorbance peak about 950 cm−1 in the outer fingerprint region was present in six groups and was absent in G2 and G6 (
Five different types with different spectra (different absorbance peaks) were obtained of the genotypes VanA, VanB and VanA plus B, one group of VanB (1 isolate), one of VanA plus B (1 isolate) and 3 of VanA (4 isolates) as shown in Figures 2-4 and
Sample No. | Disk diffusion results/zone diameter (mm) | Minimum inhibitory concentration (µg/m1) | Genotypes | ||||
---|---|---|---|---|---|---|---|
Ampicillin | Teicoplanin. | Vancomycin | Ampicillin | Teicoplanin. | Vancomycin | ||
E. faecalis ATCC 51299 | 20 | 16 | 15 | <2.0 | <2.0 | 32 | |
48198 | 0 | 10 | 0 | 256 | 256 | 256 | VanA |
4898 | 0 | 0 | 0 | 16 | 128 | 256 | VanA |
41720 | 8 | 20 | 17 | 32 | 16 | 32 | VanA |
44932 | 22 | 18 | 17 | <2.0 | <2.0 | <2.0 | |
44404 | 27 | 21 | 19 | <2.0 | <2.0 | <2.0 | VanB |
45805 | 22 | 20 | 18 | <2.0 | <2.0 | <2.0 | |
43191 | 25 | 19 | 17 | <2.0 | <2.0 | <2.0 | |
48278 | 0 | 0 | 0 | 256 | 256 | 256 | VanA+B |
45511 | 0 | 12 | 0 | 256 | 256 | 256 | VanA |
44218 | 0 | 20 | 18 | 64 | <2.0 | <2.0 | |
41509 | 24 | 19 | 18 | <2.0 | <2.0 | <2.0 | |
47892 | 22 | 17 | 17 | <2.0 | <2.0 | <2.0 | |
47808 | 21 | 19 | 17 | <2.0 | <2.0 | <2.0 | |
47781 | 22 | 20 | 19 | <2.0 | <2.0 | <2.0 | |
45416 | 22 | 19 | 17 | <2.0 | <2.0 | <2.0 | |
41838 | 0 | 19 | 20 | 256 | <2.0 | <2.0 | |
45148 | 22 | 18 | 17 | <2.0 | <2.0 | <2.0 | |
47891 | 23 | 19 | 17 | <2.0 | <2.0 | <2.0 | |
44083 | 28 | 22 | 20 | <2.0 | <2.0 | <2.0 |
Phenotype | Sample No | Absorbance Peaks in the Range (1000 - 500 cm−1) | ||||
---|---|---|---|---|---|---|
ATCC 51299 VanB | ATCC 51299 | 950.11 | 829.56 | 738.06 | 725.72 | 610.87 |
Van B G1 | 44404 | 949.94 | 828.92 | 738.01 | 727 | 610.22 |
VanA G2 | 41720 4898 | - - | 889.77 889.28 | 738.38 738.25 | 618.19 611.17 | - 541.28 |
VanA G3 | 48198 | 950.36 | 829.02 | 738.15 | 610.66 | 508.49 |
VanA G4 | 45511 | 950.06 | 830.23 | 724.74 | - | 518.32 |
VanA+B G5 | 48278 | 950.66 | 828.69 | 738.15 | 610.68 | - |
Phenotype | Sample No. | Absorbance Peaks in the Range (1000 - 500 cm−1) | ||||
---|---|---|---|---|---|---|
G6 | 43191 | 545.06 | 611.44 | 738.47 | 892.21 | - |
G6 | 45148 | 544.57 | 610.42 | 738.55 | 897.94 | - |
G6 | 47891 | - | 611.03 | 738.30 | 890.80 | - |
G6 | 44218 | - | 612.08 | 738.43 | 889.16 | - |
G7 | 47892 | 511.86 | - | 724.75 | 830.48 | 949.57 |
G7 | 44932 | 520.05 | - | 724.82 | 830.36 | 949.94 |
G7 | 44083 | 514.74 | - | 724.85 | 830.36 | 949.87 |
G7 | 47808 | 509.83 | - | 724.88 | 830.23 | 950.33 |
G7 | 41838 | 518.42 | - | 724.77 | 830.48 | 950.14 |
G8 | 45805 | 523.09 | 610.31 | 738.16 | 829.14 | 950.21 |
G8 | 47781 | 508.54 | 610.72 | 738.23 | 828.09 | 950.82 |
G8 | 45416 | 531.7 | 610.92 | 738.18 | 828.69 | 950.62 |
G8 | 41509 | 519.74 | 615.90 | 738.77 | 830.14 | 950.03 |
not identical to ATCC which is also VanB genotype as shown in
Enterococcus sp. is considered the second most common bacteria responsible for nosocomial infections with increasing ampicillin and vancomycin resistance [
of FTIR technique was tested by subjecting the same isolates to the test on different separated times. According to FTIR results, the 19 enterococcus isolates were divided into 8 clusters. Predominant absorbance peaks were present in all spectra, such as the peaks that arise at 725 or 738 cm−1 and it was noted that most of the groups had an absorbance peak at 830 cm−1 except for groups 2 and 6, the absorbance peak was at 889 cm−1. Another noticeable absorbance peak at 950 cm−1 was present in six of the eight groups and absent in groups 2 and 6. This could be explained that groups 2 and 6 might be of different species. We suggest that further biochemical investigation might be needed for confirmed identification.
FTIR spectroscopy studies to bacteria employ traditional windows such as Zinc selenide (ZnSe) in preparing the adhesive culture [
In
FTIR spectroscopy is a methodology that fingerprints the whole cell and is able to detect subtle compositional changes that cannot be revealed by conventional phenotypic or even genotypic methods. Contrary, antibiotic susceptibility testing was not discriminative enough when compared to FTIR. The later technique was used in this study to type Enterococcus clinical isolates and has proven very useful. It is easy to carry and allows the analysis of large number of strains, although it requires a high degree of standardization and complex data processing. In conclusion, certain physical and chemical properties do exist that can be determined by other means to study different molecules and organic compounds constituting part of the cell structure. This observation indicates that Enterococci bacteria species have organic compounds which can be used to identify its type and it is believed that FTIR has great potential in such clinical microbiology application.