Evaluation of Resistance Determinants and Clonal Relationship of Extended-Spectrum Beta-Lactamase Positive Gram-Negative Bacterial Strains by PCR and Raman Spectrophotometry

Background: ESBLs are enzymes which are capable of hydrolysing penicillins, broad-spectrum cephalosporins and monobactams. They are generally derived from TEM and SHV-type enzymes. Materials and methods: We investigated the presence of TEM, SHV, CTX-M, OXA and IBC beta-lactamase genes by PCR in 300 E. coli, 130 Klebsiella spp., 100 P. aeruginosa ve 23 Enterobacter spp. isolated from various clinical specimens recovered from inpatient and outpatients. Additionally, epidemiological relationships were evaluated by Raman spectromicroscopy method. Results: According to the PCR results; blaTEM (49.3%) and blaCTX-M (52.6%) were found at high rates, but blaIBC was not detected in any isolates. When PCR test results were evaluated according to the origin of bacteria, the highest rates of blaTEM (69.3%) ve blaCTX-M (79.3%) were detected in E. coli isolates, while the lowest rate (1%) was found in P. aeruginosa. With raman spectromicroscopy, it was identified 9 cluster in E. coli, 6 cluster in K. pneumoniae and K. oxytoca, 11 cluster in P. aeruginosa and 3 cluster in E. Cloacae Conclusion: Although we obtained valuable results in the comparison of the raman spectrums in positive for resistance genes, there is a great necessity for standardisation of the studies prior to the usage of the test routinely.

TEM-beta-lactamases are the most common type of enzyme among Enterobacteriaceae and were also detected in P. aeruginosa [12][13][14]. While TEM-1 and TEM-2 could hydrolyse penicillin and first generation cephalosporins. SHV-1 beta-lactamases, generally encoded chromosomally in most strains, were first detected among K. pneumoniae and spread to other Enterobacteriacea species [13,15]. SHV-1 is resistant to ampicillin, ticarcillin and piperacillin but has no effect on oximinocephalosporins. CTX-M beta lactamase originates 002 Advances in Biotechnology & Microbiology from chromosomal AmpC enzymes of Klyuvera ascorbata due to horizontal gene transfer and mutations and it shows 40% similarity with TEM and SHV enzymes. These are more active against cefotaxime and ceftriaxone than ceftazidime, even though point mutations can increase the activity towards ceftazidime [16]. Recently, it is reported that CTX-M15 is the most common enzyme globally. While most ESBLs are detected among Enterobacteriaceae, OXA types are commonly found in P. aeruginosa [13,14].
These are commonly spread with plasmid and transposon yielding resistance to aminopenicillin and ureidopenicillin. They have the ability to hydrolize oxacillin, cloxacillin and methicillin [13,14]. Inhibitor-resistant beta-lactamases (IRT), a variant due to mutations of SHV and TEM, has no ESBL activity and can not hydrolyze third generation cephalosporins and they are resistant to SAM and AMC but susceptible to TPZ [13]. IRT was commonly found among E. coli but also reported among other Enterobacteriaceae [14,15]. IBC-1, integron related Class A wide spectrum beta-lactamases, is highly resistant to ceftazidime, intermediate susceptible to cefotaxime, cefepime, aztreonam and less susceptible to clavulanic acid and piperacillin-tazobactam compared to other wide spectrum beta-lactamases [15,17]. It was first detected in E. cloacae and then in E. coli [11,18,19]. Recently, IBC-2, a variant of IBC-1 has been detected among P. aeruginosa strains. Additionally, non-TEM and non-SHV ESBLs such as PER, VEB, GES, TLA were reported [13].
New approaches of bacterial identification have been considered recently for the rapid and accurate identification of bacteria. Vibrational spectroscopic techniques, infrared (IR) and Raman spectroscopy (RS), are commonly used in chemistry, since vibrational information is specific to the chemical bonds and symmetry of molecules. The mechanism of these systems is based on an intense beam of laser in the visible or infrared or ultraviolet region focused on the sample and detecting the scattered beam to get information about the vibration modes of the sample molecules. RS is a powerful molecular fingerprinting technique by which the molecule and bacteria can be identified through the interaction of coherent light and the sample's molecules. It has recently gained popularity as an attractive approach for the biochemical characterization, rapid identification, and accurate classification of a wide range of bacterial species and strains [20][21][22][23][24]. This method is in clinical use in some advanced microbiology laboratories in recent years with the advantages of the ease for sample preparation, faster test results, reproducibility and higher discrimination power compared to other phenotypic and genotypic methods.
Studies have demonstrated that Raman spectra generated from bacterial and fungal colonies give sufficient information to identify and differentiate microorganisms and also for biofilm detection [18,[20][21][22]. Raman signals obtained from bacterial samples suffer from weakness and a huge background. Surface enhanced Raman spectroscopy (SERS) is the most common and widely used way to amplify the weak Raman signal is to attach the sample to a metallic rough surface [18,20,23].
In this study, we aimed to determine the frequencies of beta-lactamase genes, TEM, SHV, CTX-M, OXA ve IBC by PCR and epidemiological clonal relationship by Raman spectromicroscopy among ESBL-positive E. coli, K. pneumoniae, Enterobacter spp. and P. aeruginosa recovered from various clinical specimens.

Identification of resistance genes by PCR
DNA templates for polymerase chain reaction (PCR) were obtained from isolates on nutrient agar. DNA extraction was performed by phenol:chloroform:isoamyl method [25]. The ESBL genes (bla IBC , bla OXA , bla CTX-M , bla TEM and bla SHV ) were identified by PCR as previously described [26]. Primers used in this study How to cite this article: Gülnur [26].

Raman Spectromicroscopy
Preparation of bacterial cells: All strains were cultured in a nutrient broth at 37°C overnight, then 1 µL of the suspension was streaked on nutrient agar and incubated at 37 °C for 24 hours. Single colonies were harvested from the plates using an inoculating loop and suspended in 1.5 ml of deionized water. These aliquots were centrifuged at 5000 g for 3 min. The supernatant was discarded and pellet was used for the test. 10 µL of each of the suspensions was transferred to a lowfluorescence quartz microscope slide and allowed to dry at room temperature.

Preparation of the solutions: Delta Nu Examiner Raman
Microscopy System (Deltanu Inc., Laramie, WY) was used in the analysis of bacterial strains. Parameters were set on the programme of 20x objective, 30 µm laser spot area, 220 mW laser power and 60 seconds of data collection time. Spectrums were obtained between 200-2000 cm -1 range. CTAB-stable golden nanorods were used in this study. Gold nanorods were synthesized with the minor revisions in the technique of the nucleus magnification. Nucleus solution was prepared with 7.5 ml CTAB and 250 µl AuCl 4 . Afterwards, 600 µl of NaBH 4 solution that was previously prepared in the ice bath was added to the mixture quickly and left in room temperature for 30 minutes prior to use. Magnification solution was prepared using 4.75 ml CTAB, 500 µl AuCl 4 and 60 µl AgNO 3 and 100 µl ascorbic acid. 10 µL of nucleus solution was added to magnification mixture and left in room temperature for an hour prior to use.

Analysis and SERS measurement
Aluminium surface of the TLC paper was used for SERS measurement surface. SERS spectrum for each bacterial strain was obtained by dropping 2 µl of bacterial suspension onto TLC paper and interacting to the paper with 1 µl of gold nanorod solution. Median values of the signals from at least three different analysis area were evauated. Data analysis were performed by MATLAB version 7.1 (Mathworks, Natick, MA).

Statistical analyses
Cluster analysis of the spectral sets were performed by SpectraCell RA software (River Diagnostics). MATLAB version 7.1 (The MathWorks, USA) programme was used for histogram and correlation matrix. The similarities between spectrums were calculated using Pearson correlation coefficient analysis (R2) and was multiplied by 100 to express percentage. Data were analyzed using SPSS software 15.0 (SPSS, Inc., Chicago, IL, USA). Comparisons of categorical variables were done using Chisquare tests, although Fisher's exact test was used when data were sparse. Significance was set at p < 0.05 using two-sided comparisons.

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with 31.4% of the strains. blaSHV, blaTEM, blaCTX-M gene loci was found in 1% of the P. aeruginosa strains but blaOXA gene was not detected. blaTEM positivity was 30.4% for Enterobacter spp. and blaSHV, blaCTX-M and blaOXA gene loci was not detected. Overall the E. coli isolates tested (n=300), 157 was positive for blaTEM+blaCTX-M gene and eight was positive for SHV+TEM+CTX-M. In a K. pneumonae isolate recovered from aspirate (0.8%), all four gene loci except IBC were detected. Presence and distribution of blaSHV, blaTEM, blaCTX-M, blaOXA and blaIBC gene was shown on Table II and III.      In the second part of the study, subtype analysis of the strains positive for any of beta-lactamase gene loci by PCR were evaluated using Raman spectromicroscopy with five different concentration. Five μL of bacterial suspension was added to 1X nanoparticule suspansion prepared as 100 μL, 125 μL, 150 μL, 175 μL, 200 μL. Raman spectromicroscopy did not show marked difference in the spectrum among the prepared solutions in some strains. As shown in Figure 2, it is observed that the best bacterial concentration was 5 μL bacteria added to 100 μL of nanoparticule suspension. Same results were achieved in the following four repeated tests performed with same samples and same test conditions. Concentration of 5 μL bacterial suspension to 100 μL of nanoparticule suspension was used for the evaluation of the strains positive for any of blaSHV, blaTEM, bla CTX-M and bla OXA gene loci. Weak signal was detected in E120 strain that any of the gene loci was not detected. A strong and discriminative spectrum was detected among E. coli E113, E. coli E66 and K. pneumoniae K9 strains that were positive for blaSHV, blaTEM and blaCTX-M by PCR. All bacterial strains showed similar results in this evaluation (Figure 3-6).

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Bacterial typing with Raman spectrum revealed that nine cluster among E. coli strains, six cluster among Klebsiella spp., 11 cluster among P. aeruginosa and three cluster among E. cloacae were detected. Cluster analysis was not performed for E. aerogenes strains due to the low number of the strains.

Discussion
Extended-spectrum beta-lactamase producing and multidrug-resistant Gram-negative bacteria related infections are one of the major growing concerns worldwide. Thus, rapid and accurate detection of resistance mechanism and determinants has critical importance in the control of the infections and determining the treatment options [2].
In this study, a total of 553 ESBL-positive Gram-negative bacteria recovered from various infection sites were evaluated for beta-lactamase gene presence including bla IBC, bla TEM, bla SHV, bla CTX-M by PCR and raman spectroscopy. Among all isolates tested blaTEM 49.3% and blaCTX-M 52.6% gene loci was detected but bla IBC presence was not detected. Evaluation of the gene loci presence by bacterial strains revealed that bla TEM (69.3%) and bla CTX-M (79.3%) genes were higher among E. coli and blaSHV (41.5%) and bla TEM (44.6%) gene was higher among Klebsiella strains. While 1% of P. aeruginosa strains were positive for blaSHV, bla TEM, bla CTX-M genes, bla OXA was not detected. The most co-existence of the gene loci was blaTEM and bla CTX-M with 31.4% of the strains. Of 300 E. coli strain tested 165 were positive for bla TEM and bla CTX-M.
In a study conducted by Paterson et al. [11] including 455 K. pneumoniae strains recovered from 12 hospitals from seven countries were evaluated for ESBL presence and among ESBLpositive strains 67.1% were positive for SHV, 16.4% for TEM, 23.3% for CTX-M. This is worrisome, especially in Turkey where ESBL prevalence is very high [6,25]. In the past decade blaCTX-M gene has replaced blaSHV and blaTEM genes in Canada, Europe and Asia as the most common ESBL type in these bacteria similar with our findings. The CTX-M beta-lactamases are now widespread in both nosocomial and community-acquired pathogens. The blaTEM gene has a high frequency compared to bla SHV gene which is similar to our finding.
Raman spectroscopy is a new method in the determination and typing of infectious microorganisms. Although it has been used for a long time for the chemical characterization of different materials, it has just lately been applied to the study of biological samples in order to provide a rapid identification and discrimination of pathogenic organisms [18,[20][21][22]. Data concerning issue is scarce worldwide. In our study, epidemiological typing with Raman spectrums was performed for the strains found to be positive for any of the five gene loci and found that E. coli has nine cluster, K. pneumoniae and K. oxytoca has six cluster, P.aeruginasa has eleven cluster and E. cloacae has three clusters. Due to the low number of the strains E. aerogenes strains were not included for typing. Comparison of the raman spectrums of the strains showed that higher spectrums were detected among strains positive for resistance genes. There is a great necessity for standardisation of the studies prior to the usage of the test routinely. Although, this method gives valuable information, it doesn't seem that this method could be alternative to PFGE, gold standart for epidemiologial typing and it should be reevaluated with a reference method. Further studies should be conducted for better understanding and standardised the method itself.
The use of some first line treatment antibiotics such as penicillin and trimethoprim/sulfamethoxazole seem inappropriate. Antibiotics resistance surveillance and the determination of molecular characteristics of ESBL isolates are primordial to ensure the judicious use of antimicrobial drugs. The prevalence of beta-lactamase producing isolates and their isolation from life-threatening infections, is increasing at an alarming rate worldwide. It was shown in this study that betalactamase producing E. coli strains are an emerging threat in hospitals and should be supervised by implementation of timely identification and strict isolation methods that will help reduce their severe outcomes and mortality rate in this patients. In conclusion, this study has confirmed the potential of Raman spectroscopy to identify bacteria.