|Year : 2017 | Volume
| Issue : 1 | Page : 61-68
Characterisation of antimicrobial resistance in Salmonellae during 2014–2015 from four centres across India: An ICMR antimicrobial resistance surveillance network report
Sushila Dahiya1, Priyanka Sharma1, Bhavana Kumari1, Sangeeta Pandey1, Rooma Malik1, Neelam Manral1, Balaji Veeraraghavan2, Agila Kumari Pragasam2, Pallab Ray3, Vikas Gautam3, Sujata Sistla4, Subhash Chandra Parija4, Kamini Walia5, Vinod Ohri5, Bimal Kumar Das1, Seema Sood1, Arti Kapil1
1 Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India
2 Department of Microbiology, Christian Medical College, Vellore, Tamil Nadu, India
3 Department of Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
4 Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India
5 ICMR, New Delhi, India
|Date of Web Publication||16-Mar-2017|
Department of Microbiology, All India Institute of Medical Sciences, New Delhi
Source of Support: None, Conflict of Interest: None
Purpose: The main purpose of this study was to establish 'Antimicrobial Resistance Surveillance Network' in India and to monitor the antimicrobial susceptibility profile of clinical isolates to establish a national network across the country for monitoring antimicrobial resistance in Salmonella. Materials and Methods: This study was conducted at All India Institute of Medical Sciences, nodal centre with clinical isolates of Salmonellae collected from four centres across India, which included Christian Medical College, Vellore; Postgraduate Institute of Medical Education and Research, Chandigarh and Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry. Total 20% of the selected strains from each centre were characterised for molecular studies which included molecular mechanism of fluoroquinolones resistance and multiple locus sequence type. Results: A total of 622 Salmonellae were received from all centres during January 2014 to December 2015. Out of these 622 isolates, 380 were Salmonella Typhi, 162 were Salmonella Paratyphi A and 7 were S. Paratyphi B isolated from blood and 73 were other Salmonella serotypes. Multiple drug resistance (resistant to ampicillin, chloramphenicol and co-trimoxazole) was less than 3% in S. Typhi. In S. Paratyphi A, chloramphenicol and co-trimoxazole susceptibility was 100% and 99%, respectively, whereas ampicillin susceptibility was 86% (139/161). Ciprofloxacin and nalidixic acid susceptibility was 15% (24/162) and 1% (2/162) from all centres. S. Paratyphi B was isolated from 7 patients. All isolates were third-generation cephalosporin sensitive. The most common mutations found were at codon 83 and at codon 87. We did not find any mutation in acrR gene. Efflux pump and qnr genes were not found in any isolate tested. All 86 S. Typhi isolates clustered into two sequence types - ST1 and ST2. Out of these 86 isolates, 70 S. Typhi were ST1 and 16 were ST2. All S. Paratyphi A was clustered in ST85 and ST129 on the basis of mutation in sucA gene. Out of 27 S. Paratyphi A, 13 were grouped into ST85 and 14 were grouped into ST129. Conclusions: Enteric fever is one such infection which poses challenges in antimicrobial resistance. Hence, continuous surveillance is important to track bacterial resistance and to treat infections in a cost-effective manner.
Keywords: Antimicrobial Resistance Surveillance Network, efflux pump, enteric fever, multiple locus sequence type, quinolone resistance determining region, Salmonella
|How to cite this article:|
Dahiya S, Sharma P, Kumari B, Pandey S, Malik R, Manral N, Veeraraghavan B, Pragasam AK, Ray P, Gautam V, Sistla S, Parija SC, Walia K, Ohri V, Das BK, Sood S, Kapil A. Characterisation of antimicrobial resistance in Salmonellae during 2014–2015 from four centres across India: An ICMR antimicrobial resistance surveillance network report. Indian J Med Microbiol 2017;35:61-8
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Dahiya S, Sharma P, Kumari B, Pandey S, Malik R, Manral N, Veeraraghavan B, Pragasam AK, Ray P, Gautam V, Sistla S, Parija SC, Walia K, Ohri V, Das BK, Sood S, Kapil A. Characterisation of antimicrobial resistance in Salmonellae during 2014–2015 from four centres across India: An ICMR antimicrobial resistance surveillance network report. Indian J Med Microbiol [serial online] 2017 [cited 2017 Mar 29];35:61-8. Available from: http://www.ijmm.org/text.asp?2017/35/1/61/202344
| ~ Introduction|| |
Enteric fever is a major public health problem in many parts of the world without adequate access to safe water and sanitation and is responsible for high morbidity despite available antibiotics.Salmonella enterica serotype Typhi(S. Typhi) is the most common causative agent of enteric fever followed by S. enterica serotype Paratyphi A (S. Paratyphi A). Although S. Paratyphi B predominates in Europe, S. Paratyphi C is rare and is seen only in the Far East.
The incidence of enteric fever is high in Southcentral and South-East Asia. The recent estimates of global incidence of enteric fever are between 11.9 million and 26.9 million cases per year., Case fatality rate remains 1% ranging between 129,000 and 161,000 typhoid deaths annually. A community-based study from India reported an incidence rate of 27.3/1000 person-years in the under 5-year age group which was also a significant cause of morbidity in children. The peak incidence of enteric fever occurs between April and June followed by July-September.
The major challenge in enteric fever at present is the increase in antimicrobial resistance in S. Typhi and S. Paratyphi A, especially to fluoroquinolones. The reports on ciprofloxacin resistance started to appear soon after its clinical use. The regular revisions of Clinical Laboratory Standards Institute (CLSI) guidelines in the interpretative criteria in 2011 and addition of new fluoroquinolones in 2015 and 2016 indicate the urgency and need to revise breakpoints to optimise the dose of fluoroquinolones and use this drug effectively in susceptible isolates. As more fluoroquinolones are being added in the guidelines, pefloxacin has been recommended as a surrogate marker for all fluoroquinolones to facilitate the testing. At present, ceftriaxone is the best available drug., The minimum inhibitory concentration (MIC) for ceftriaxone shows creeping MIC trend over the years with recent reports of clinical failure to ceftriaxone., However, now resistance to ceftriaxone has also been reported., The other antibiotic being used is azithromycin which is recommended for uncomplicated cases though adequate clinical studies and PK/PD parameters in typhoid fever are lacking.
The study of mechanism of resistance to fluoroquinolones at molecular level suggests DNA gyrases and topoisomerase gene mutations to be the common mechanism. Efflux pump has not been documented to be responsible for resistance except for isolated reports. The molecular characterisation also helps in understanding the spread of bacteria over a geographical area and multiple locus sequence type (MLST) is one such method that can be used for the population structure study and detecting genetically related clones.
Therefore, an estimate of the regional burden of Salmonella with antimicrobial resistance and phylogenetic analysis is needed to inform and strengthen efforts to prevent the emergence of resistance and decide antibiotic policy for enteric fever in India. Indian Council of Medical Research conducted a surveillance study across four centres in India which included All India Institute of Medical Sciences, New Delhi (AIIMS), Christian Medical College, Vellore (CMC), Postgraduate Institute of Medical Education and Research, Chandigarh (PGIMER) and Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry (JIPMER). The main objective of this study was to (1) establish 'Antimicrobial Resistance Surveillance Network' in India, (2) to monitor the antimicrobial susceptibility profile of clinical isolates and (3) to establish a national network across the country to undertake surveillance and monitoring of antimicrobial resistance in Salmonellae which is reported in the present study.
| ~ Materials and Methods|| |
This study was conducted at AIIMS nodal centre with clinical isolates of Salmonellae collected from four centres across India, which included AIIMS, New Delhi; CMC, Vellore; PGIMER, Chandigarh and JIPMER, Puducherry. Standard operative protocols for identification and antimicrobial resistance testing were developed by ICMR with all the participating centres for uniformity across the centres. Updated CLSI guidelines for susceptibility testing of Salmonella were used.
For quality control, all the Salmonellae isolates from 4 centres were retested at the AIIMS nodal centre. AIIMS nodal centre was participating in IAMM EQAS which is a national proficiency testing in microbiology in India. For internal QC, Escherichia coli ATCC 25922 was used as reference strain for QC of antibiotic discs and Enterococcus faecalis ATCC 29212 for co-trimoxazole disc. ATCC Ty2 Salmonella was used as reference strains for molecular studies.
Antimicrobial susceptibility testing
Antimicrobial susceptibility was determined by Kirby-Bauer disc diffusion method according to the CLSI guidelines 2014 and 2015 for amoxicillin (10 µg), co-trimoxazole (1.25/23.75 µg), ciprofloxacin (5 µg), nalidixic acid (30 µg), chloramphenicol (30 µg), ceftriaxone (30 µg) and cefixime (5 µg).
Strains showing multiple resistant to ampicillin, chloramphenicol and co-trimoxazole were defined as multiple drug resistant (MDR) while nalidixic acid resistant (NAR) and nalidixic acid sensitive were defined based on susceptibility to nalidixic acid.
Minimum inhibitory concentration by E-test
MIC for ciprofloxacin and ceftriaxone was determined by E-test and E. coli ATCC 25922 was used as reference strain.
Extended-spectrum beta-lactamase screening
For ceftriaxone or cefixime-resistant isolates, screening for extended-spectrum beta-lactamases (ESBLs) enzymes was done according to the CLSI guidelines (2012).
As per the predefined protocol by ICMR, 20% of the selected strains from each centre were characterised for molecular studies which included molecular mechanism of fluoroquinolone resistance and MLST.
DNA extraction was done by commercially available Qiamp DNA extraction kit (QIAamp DNA Mini Kit; Qiagen, Hilden, Germany). DNA extraction yielded about 2 μg DNA (elution volume was 100 μl). Hence, 100 ng of DNA (per 5 μl) was used in the polymerase chain reaction (PCR) as template.
Mutations in DNA gyrase
The quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC and parE from Salmonella with decreased susceptibility or resistant to ciprofloxacin were amplified by PCR, and sequencing was done as described previously from our laboratory. PCR was performed on genomic DNA of each isolates in a total volume of 50 μl, which contained 5 μl of supernatant, 5 pmol of each primer, 200 μM deoxynucleoside triphosphates and 2.5 U of Taq polymerase. PCR primers and conditions are given in [Table 1].
|Table 1: Details of primer and PCR conditions for the amplification of QRDR and QNR genes used for fluoroquinolones resistance study|
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To study the role of efflux pump, acr R Regulator gene was amplified. The forward primer: 5'-GGTCCTTAAACCCATTGCTG-3'and reverse primer: 5'-CAGAATAGCGACACAGAAA-3' were used to obtain a 816 bp amplicon. The PCR was carried out at the following reaction conditions: initial PCR activation for 5 min at 94°C, enrichment cycling for 30 cycles of (15 s at 94°C, 60 s at 57°C and 60 s at 72°C) and final extension for 10 min at 72°C. Sequencing of the gene product was done by method mentioned below.
Ciprofloxacin-resistant strains were screened by PCR for plasmid-mediated quinolone resistance determinants, namely, qnr alleles (qnrB and qnrS). As qnrA has not been reported in Salmonella, we screened only for the presence of qnrB and qnrS genes.
Briefly, plasmid-mediated quinolone resistance was screened by multiplex PCR amplification of qnrB, and qnrS using primers previously described. All four primers were added to the template. The PCR conditions were 95°C for 30 s, 55°C for 1 m and 72°C for 30 s cycled 35 times [Table 1]. Clinical isolates from other species were screened for the presence of qnr genes and qnrB and qnrS were found in Klebsiella isolates. These positive isolates were used as positive control.
Multiple locus sequence type
MLST was done in 86 S. Typhi and 27 S. Paratyphi A strains. Seven housekeeping genes of known function and chromosome position, thrA (aspartokinase + homogenise dehydrogenase), purE (phosphoribosylaminoimidazole carboxylase), sucA (alpha-ketoglutarate dehydrogenase), hisD (histidinol dehydrogenase), aroC (chorismate synthase), hemD (uroporphyrinogen III cosynthase) and dnaN (DNA polymerase III beta subunit) were used for MLST. Primer and PCR conditions are given in [Table 2]., DNA extraction was done by commercially available kit as described above. The PCR was performed in a final reaction volume of 50 μl containing 5 μl of 10X polymerase buffer and 2.65U of Taq DNA polymerase. The PCR-amplified DNA segment was electrophoresed in 1.5% (w/v) agarose gel (Life Technologies, GibcoBRL, Scotland) prepared in 0.5X Tris-borate ethylenediaminetetraacetic acid buffer (Sigma-Aldrich Pvt. Ltd., India), along with the DNA molecular weight marker (100 bp DNA ladder) (Banglore Genie Pvt. Ltd., India). The PCR product was observed after staining agarose gel with ethidium bromide (0.5 μg/ml) using a 'ChemiImager Ready' gel documentation system (Alpha Innotech Corporation, California, USA) and the gels were photographed using Gel Doc™ (Bio-Rad, Hercules, Calif, USA). Sequencing was done by the method described below.
Sequencing was carried out by the dideoxynucleotide chain-termination method, using an automated DNA sequencer ABI PRISM ® 310 Genetic Analyzer (Applied Biosystems, USA) using AmpliTaq Gold DNA polymerase (Applied Biosystems, USA) which is a modified form of AmpliTaq DNA polymerase.
Sequence analysis for quinolone resistance
Forward and reverse DNA sequences were assembled, trimmed, edited and analysed for each gene fragment. Reference sequences for all QRDRs (gyrA, gyrB, parC and parE) and acrR were obtained from http://www.ncbi.nlm.nih.gov/gene and compared with the sequence of S. Typhi strain CT18 (accession no. AL627274). Sequence chromatograph files were analysed using BioEdit version 5.0.918 to resolve nucleotide ambiguities. Global alignment of sequences was done using the software Clustal X version 1.8 and GeneDoc version 2.1.02.,
For phylogenetic relationships amongst S. Typhi and S. Paratyphi A isolates, forward and reverse DNA sequences were assembled, trimmed, edited and analysed for each gene fragment.
Briefly, multiple alignments were done using GeneDoc Multiple sequence alignment editor and shading utility version 2.6.00213 and Clustal X 1.8114. ST (sequence type) was assigned based on the allelic profile. The seven housekeeping genes were concatenated for all isolates and using the unweighted pair group method with arithmetic averages using MEGA6 phylogenetic tree was constructed.
Salmonellae Agona, Salmonellae montevideo, Salmonellae newport, Salmonellae enteritidis, Salmonellae dublin, S. Agona, Salmonellae typhimurium, Salmonellae saintpaul, S. Paratyphi B, S. Paratyphi A and Salmonellae heidelberg were taken as outgroup.
| ~ Results|| |
A total of 622 Salmonellae were received from all centres during January 2014 to December 2015. All the isolates were obtained from various clinical samples, for example, blood, pus, urine and sterile fluid. Out of these 622 Salmonella, 380 were S. Typhi, 162 were S. Paratyphi A and 7 were S. Paratyphi B isolated from blood and 73 were other Salmonella serotypes [Table 3], [Figure 1]a and [Figure 1]b.
|Figure 1: (a) Total distribution of Salmonella Typhi, Paratyphi A and Paratyphi B according to isolates received from each centre. (b) Distribution of non-typhoidal Salmonella from each centre.|
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We found S. Typhi susceptibility to chloramphenicol and co-trimoxazole was 97% (369/380) and 95% (360/380), respectively, from all centres, whereas ampicillin susceptibility was 86% (329/380). When sensitivity was compared centre wise, we found that susceptibility was 100% for chloramphenicol and co-trimoxazole from Chandigarh and 100% for chloramphenicol from Puducherry. Ampicillin susceptibility was also high in Chandigarh i.e., 96% (206/215).
Multiple drug resistance (Resistant to ampicillin, chloramphenicol and co-trimoxazole) was less than 3%, whereas centre wise, it was observed in 6% of S. Typhifrom AIIMS, New Delhi and CMC, Vellore.
Ciprofloxacin susceptibility was 20% (77/380) in all centres, whereas nalidixic acid susceptibility was 24% (91/380) [Table 4a].
In S. Paratyphi A, chloramphenicol and co-trimoxazole susceptibility was 100% and 99%, respectively, whereas ampicillin susceptibility was 86% (139/161). Ciprofloxacin and nalidixic acid susceptibility was 15% (24/162) and 1% (2/162) from all centres [Table 4b].
|Table 4b :Antibiogram of S. enterica serovar Paratyphi A. All Centers (% susceptible)|
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Third-generation cephalosporins were 100% sensitive in S. Typhi and S. Paratyphi A, whereas a creeping increase in MIC pattern was observed for ceftriaxone from 0.032 to 0.94 in S. Typhi followed by 0.019–0.75 over the years [Table 5], [Figure 2]a and [Figure 2]b. None of the strain was found ESBL positive.
|Figure 2: (a) Minimum inhibitory concentration 50 and minimum inhibitory concentration 90 for ciprofloxacin and ceftriaxone in Salmonella Typhi. (b) Minimum inhibitory concentration 50 and minimum inhibitory concentration 90 for ciprofloxacin and ceftriaxone in Salmonella Paratyphi A.|
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S. Paratyphi B was isolated from seven patients. Out of which two were ciprofloxacin intermediate and five were NAR. All were third-generation cephalosporin sensitive but with increased MIC (0.062–0.25). All were sensitive for first-line drugs.
One S. Weltevredenand S. vein from Chandigarh were resistant to ciprofloxacin and nalidixic acid.
For the study of fluoroquinolones resistance in S. Typhi and S. Paratyphi A, sequence analysis of QRDR region of DNA gyrase and topoisomerase gene revealed the presence of a mutation in all the ciprofloxacin-resistant strains studied, whereas none of the ciprofloxacin-sensitive strain had a mutation in this region. The most common mutations found were at codon 83 where a C→T/A transition led to the substitution of phenylalanine/tyrosine for serine (Ser83→Phe) and at codon 87, (G→A/G/T transition) which results in a change of Asp87 → Asn/Gly/Tyr.
The entire acrR gene was amplified to ensure the detection of any mutation in the gene including at the hot spot. We did not found any mutation in acrR gene. Efflux pump was not responsible for ciprofloxacin resistance in S. Typhi and S. Paratyphi A studied. Qnr genes were not found in any isolate tested.
Based on concatenated sequence of six housekeeping genes, all S. Typhi isolates were uniform. However, all isolates were mutated at one site in hemD gene resulting in non-synonymous changes in the gene product. All 86 S. Typhi isolates showed monophyletic lineage [Figure 3] and clustered into two sequence types - ST1 and ST2. Out of these 86 isolates, 70 S. Typhi were ST1 and 16 were ST2. All S. Paratyphi A was clustered in ST85 and ST129 on the basis of mutation in sucA gene. Out of 27 S. Paratyphi A, 13 were grouped into ST85 and 14 were grouped into ST129.
|Figure 3: The evolutionary history was inferred using the unweighted pair group method with arithmetic averages method. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. The analysis involved 107 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 1111 positions in the final dataset. Evolutionary analyses were conducted in MEGA6.|
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| ~ Discussion|| |
For the management of typhoid fever, timely treatment with appropriate antibiotics plays a vital role in reducing mortality due to fever in addition to supportive measures. However, increasing resistance soon after the introduction of antibiotic is the major cause of concern today. Chloramphenicol was the first drug to be reported as resistant from India followed by ampicillin and co-trimoxazole in 1948 and 1972 i.e., chloramphenicol, ampicillin and co-trimoxazole called as MDR strains.,
After MDR, continuous inappropriate uses of fluoroquinolones have resulted in strains with reduced susceptibility to fluoroquinolones and clinical failure due to infection with these strains.,, This increase and spread in antibiotic resistance leads towards the limited option of treatment. Resistance to all three first-line drugs is plasmid mediated. While fluoroquinolone resistance is associated with point mutations in gyrase or topoisomerase gene.In vitro induction of mutation experiment shows accumulation of more than one mutation under selective pressure of ciprofloxacin results in higher MIC.
Following clinical failure to ciprofloxacin reports, third-generation cephalosporins such as ceftriaxone have become first line of treatment, but recently, there have been sporadic reports of clinical failure to ceftriaxone in S. Typhi and ESBL presence in non-typhoidal Salmonellae.
In this study, we observed an increase in ceftriaxone MIC over the years from 0.032 to 0.9. It may only be a matter of time before these genes encoding ESBL may crossover to the typhoidal Salmonellae and cause resistance to ceftriaxone. Third-generation cephalosporins are advised to be a reservoir drug for the treatment of MDR and ciprofloxacin-resistant strains.
In the present study, the most common causative agent was S. Typhi followed by S. Paratyphi A. In this study, only seven S. Paratyphi B were isolated from blood from Chandigarh. In case of S. Paratyphi B, there are few reports of neonatal sepsis from India. No S. Paratyphi C has been reported.
Reports of invasive nontyphoidal Salmonellae (iNTS) as a major cause of severe febrile illness in Sub-Saharan Africa are well known. However, very little is known about the incidence of iNTS in Asia, particularly from India., In this study, we found blood infections due to Salmonella species other than typhoidal Salmonella (eight from Chandigarh and two from Puducherry). One S. Weltevreden and S. Vein from Chandigarh were resistant to ciprofloxacin and nalidixic acid. While in earlier report from Asia, only one isolate was NAR.
In this study, there appears to be an increasing susceptibility to first-line drug ampicillin, chloramphenicol and co-trimoxazole i.e., S. Typhi susceptibility to chloramphenicol and co-trimoxazole was 97% and 94%, respectively. Ampicillin resistance showed an upward trend over the years in S. Typhi and S. Paratyphi A which is 86% and 87% in S. Typhi and S. Paratyphi A, respectively. However, susceptibility to all three first-line drugs for S. Paratyphi A is increasing over the years in contrast to a previous study. Ciprofloxacin resistance was 85% as compare to S. Typhi. While out of which seven S. Paratyphi B, two were ciprofloxacin intermediate and five were NAR. All were third-generation cephalosporin sensitive but with increased MIC (0.062–0.25).
We studied the mechanism of resistance and found that mutations in gyr A and par C genes responsible for ciprofloxacin resistance were present in all ciprofloxacin non-susceptible strains. No mutations were detected in gyrB and parE genes. Strains with more than one mutation in gyrA gene had a higher MIC. We did not find efflux pump and qnr genes.
In the present global scenario of changing antimicrobial resistance pattern, continuous surveillance for resistance and characterisation is important because it will inform the geographical areas which need immediate alterations with regard to the travel and deciding vaccine strategies. MLST grouped S. Typhi in ST1 and ST2 on the basis of single point mutation in hemD gene giving hemD 1 and hemD 2 allelic type and S. Paratyphi A in ST 129 and ST 85 on the basis of mutation in sucA gene giving sucA 56 and sucA 9 allelic types. This study also corroborates the result from previous studies from our region that typhoidal Salmonellae are uniform in distribution MLST database available in public domain. The reports of haplotype H58 clone associated with ciprofloxacin-resistant are circulating in endemic areas.,
The continuous attempt to track bacterial resistance is a necessity, and the need to remain ahead with alternative approaches to treat infections in a cost-effective manner is a research priority in public health. Enteric fever is one such infection which poses challenges and characterising S. Typhi and Paratyphi A is relevant in medical science in the present global scenario.
Enteric fever is still a major public health problem which poses challenges in antimicrobial resistance with changing pattern over time. Hence, continuous surveillance is important to track bacterial resistance and to treat infections in a cost-effective manner. There is a need for the alternate approaches to treat infections.
Financial support and sponsorship
This study was financially supported by Indian Council of Medical Research.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4a], [Table 4b], [Table 5]