Escherichia coli in respiratory tract infections: Evaluating antimicrobial resistance and prevalence of fimA, neuC and iutA virulence genes

Gene Reports 18 (2020) 100576

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Escherichia coli in respiratory tract infections: Evaluating antimicrobial resistance and prevalence of fimA, neuC and iutA virulence genes

T

⁎

Shukla Promitea,b, , Sajal K. Sahac a

Department of Microbiology, University of Dhaka, Bangladesh Department of Infection, Immunity and Human Disease, University of Leeds, United Kingdom c Department of Clinical Pharmacy and Pharmacology, University of Dhaka, Bangladesh b

A R T I C LE I N FO

A B S T R A C T

Keywords: Antimicrobial resistance Virulence genes Escherichia coli RTIs

Optimising the treatment of respiratory tract infections (RTIs) caused by multidrug-resistant (MDR) Escherichia coli has become challenging with existing antibiotic options. E. coli pathogens have various virulence factors that determine their pathogenesis and antimicrobial resistance (AMR) mechanisms. The evidence of virulence genes harboured by E. coli in human RTIs yet remains unexplored. This study aimed to investigate the presence of fimA, neuC and iutA virulence genes encoding type 1 fimbriae, capsular polysaccharide, and siderophores respectively and AMR pattern of E. coli isolates conferring RTIs. Fifty E. coli isolates were confirmed by culture and biochemical tests. Virulence genes were identified by PCR method. AMR was assessed by disc diffusion method. The prevalence of fimA and neuC was 44%, and 24% respectively though no iutA was found. The frequency distribution of fimA and neuC genes was 42.8% and 25% in upper RTIs and 45.4% and 22.7% in lower RTIs respectively but had no statistically significant difference (fimA, p = 0.854; neuC, p = 0.851) when compared between URTIs and LRTIs. All (100%) E. coli isolates were MDR. Of 50 isolates, highest resistance was observed against amoxicillin (72%) followed by ampicillin (66%), cefixime (56%), sulfamethoxazole/trimethoprim (54%), ciprofloxacin (48%), nalidixic acid (32%) amikacin (28%) and gentamycin (24%). No E. coli including either fimA or neuC positive, was resistant to colistin, imipenem and meropenem antibiotics. This is the first report on virulence-associated genes and MDR among E. coli isolates recovered from RTIs in Bangladesh. Reduced susceptibility of MDR E. coli to most of the antibiotics in RTIs is alarming. Future research should confirm the association between AMR and virulence genes of E. coli isolates identified in RTIs to explain the pathogenesis of RTIs, mechanism of E. coli attaining MDR and antibiotic therapy in future.

1. Introduction Multidrug-resistant (MDR) Escherichia coli has been listed as a priority pathogen by the World Health Organization (WHO) due to emerging antimicrobial resistance (AMR) (WHO, 2017; RodrigoTroyano and Sibila, 2017). In developing countries, both hospital and community-acquired respiratory tract infections (RTIs) are linked with emerging MDR E. coli (Atia et al., 2018). RTIs are one of the leading causes of morbidity and mortality with > 50 million deaths per annum (Heron et al., 2009; Nepal et al., 2018). Besides, inappropriate use of antibiotics (O'Connor et al., 2018), increasing costs of treatment (Liu et al., 2015) and growing AMR (Ciofu et al., 2015) in RTIs are also a legitimate concern. To address these issues, molecular understanding of the cause of AMR and bacterial pathogenesis is increasingly important. The mechanism of E. coli resistance has been explained by an extended-

spectrum β-lactamases (ESBL), genetic mutations and transfer of plasmid DNA carrying resistant and virulence genes (Džidić et al., 2008). Virulence factors directly interfere with the mechanism of E. coli pathogenicity (Croxen and Finlay, 2010; Robins-Browne et al., 2016; Kuhnert et al., 2000). Schaufler et al. (2019) demonstrated that virulent and multidrug resistant pathogenic E. coli are emerging. The association of 58 virulence genes with E. coli strains has been found in urinary infections, neonatal meningitis and septicaemia (Chapman et al., 2006; Robins-Browne et al., 2016). However, the similar evidence of virulence association in RTIs still remains limited. Therefore, researching the virulence determinants of E. coli is increasingly important to understand the emergence of MDR in E. coli and addressing the complexity of RTIs treatment with antibiotics. Numerous virulence genes in E. coli demonstrated a relationship with AMR and bacterial pathogenicity

Abbreviation: AMR, antimicrobial resistance; RTIs, respiratory tract infections; MDR, multidrug-resistance ⁎ Corresponding author at: Department of Infection, Immunity and Human Disease, University of Leeds, Leeds LS2 9JT, United Kingdom. E-mail address: [email protected] (S. Promite). https://doi.org/10.1016/j.genrep.2019.100576 Received 26 October 2019; Received in revised form 23 November 2019; Accepted 5 December 2019 Available online 06 December 2019 2452-0144/ © 2019 Elsevier Inc. All rights reserved.

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(Kaczmarek et al., 2012). For instance, fimA gene is hypothesized to be an important virulence factor in urinary tract infections (UTIs) caused by MDR E. coli (Mitra et al., 2013). fimA gene encodes a virulence factor Type 1 Fimbriae (an outer membrane surface structures) that help E. coli to adhere, colonize and develop resistance by drug evasion (Zhou et al., 2019; Marc and Dho-Moulin, 1996; Klemm, 1984; Vizcarra et al., 2016). fimA is a major subunit of type 1 pilus (Puorger et al., 2011). Qrskov et al. (1982) reported the prevalence of fimA gene in 70% of pathogenic E. coli strains. Iron uptake receptor gene (iutA) is another virulence gene that helps in iron acquisition in E. coli by producing siderophores (iron chelators). Invasive strains of E. coli develop high-affinity iron-acquisition systems that take part in a competition with the host siderophores (transferrin), favour their growth in the bloodstream where iron content is low (De Lorenzo and Martinez, 1988; Chouikha et al., 2008). iutA protein is also thought to be a protective antigen that assists to develop resistance in extra-intestinal infections (Roberts et al., 1989). Karami et al. (2017) identified iutA as a virulence determinant to growing AMR in commensal and uropathogenic E. coli. Furthermore, neuC encodes UDP N-Acetylglucosamine 2-Epimerase that protects bacteria from antibiotic killing by developing capsular polysaccharide (Zapata et al., 1992; Vann et al., 2004). neuC was present among 2% of vaginal E. coli strains and 78–92% of neonatal meningitis E. coli strains (Kaczmarek et al., 2018; Bingen et al., 1997; Johnson et al., 2002; Korhonen et al., 1985; Obata-Yasuoka et al., 2002; Watt et al., 2003). The distribution of virulence genes among MDR pathogens depends on sources of samples and type of infections (Diarra et al., 2010). Prevalence of virulence genes has been investigated mostly in avian pathogenic and uropathogenic E. coli. Whereas, in human RTIs, this evidence for E. coli isolates remains unexplored. In addition, there are no published reports regarding the prevalence of virulence genes among E. coli clinical isolates in Bangladesh. This study aimed to investigate the presence of fimA, neuC and iutA virulence genes encoding type 1 fimbriae, capsular polysaccharide, and siderophores and AMR in E. coli isolates conferring RTIs in Bangladesh.

microbiology department of the University of Dhaka for analysis. To further confirm, E. coli isolates were cultured onto Nutrient agar, Blood agar and Eosin Methylene Blue (EMB) agar with incubation at 37 °C for 24 h. After incubation, E. coli appeared large, circular, greyish-white colonies on Nutrient agar plates, a green metallic sheen coloured colony on EMB plates and big, circular, moist and grey on Blood agar plates. The standard Gram Staining procedures were applied for all isolates (Coico, 2005). Additionally, the isolates were biochemically characterised by Kligler's Iron agar test, Motility–Indole–Ornithine test, Citrate utilization test, Methyl- Red and Voges- Proskauer test, Indole test, and Catalase test (Isenberg, 2004). Confirmed E. coli isolates were transferred into TSB medium with glycerol and stored at −80 o C. 2.2. Testing of antibiotic sensitivity AMR profiling of 50 E. coli isolates was performed by Kirby–Bauer Disk Diffusion Susceptibility method (Hsueh et al., 2010) using 13 different antibiotics. The disk diffusion test was performed on each E. coli isolate using Mueller-Hinton agar and standard antibiotic disks (Merck KGaA, Darmstadt, Germany). Antibiotic disks were chosen from different classes of antibiotics: penicillin- amoxicillin (30 μg) and ampicillin (30 μg); cephalosporin- cefixime (5 μg), ceftriaxone (30 μg) and cefepime (30 μg); fluoroquinolone- ciprofloxacin (5 μg) and nalidixic acid (30 μg); aminoglycosides- gentamycin (30 μg) and amikacin (30 μg); carbapenem- colistin (15 μg), imipenem (10 μg) and meropenem (10 μg); and sulfa drug-sulfamethoxazole/trimethoprim (25 μg) (Merck KGaA, Darmstadt, Germany). A standard chart and a guideline of Fluka-Sigma-Aldrich (2015) were used to determine the antibiotic sensitivity of E. coli isolates based on the diameter of each zone of inhibition (Fluka-Sigma-Aldrich, 2015; Hudzicki, 2009). The zone of inhibition was measured after 24 h. E. coli isolates were labelled as MDR if the isolates were resistant to at least three different antimicrobial agents. 2.3. DNA extraction

1.1. Research questions

E. coli grew on EMB medium was used to extract template DNA. We employed DNA extraction procedure as described in Kakian et al. (2019). The 4–5 colonies were placed into Eppendorf tubes having 1000 μl of Tris buffer. Eppendorf tubes were then heated for 15 min in a water bath at 95 °C. After heating, tubes were centrifuged for 10 min at 14,000 rpm (Kakian et al., 2019). Cell debris was pelleted at the bottom and the supernatant contained the DNA. This was used as a template DNA for subsequent PCR.

This study explores the below specific research questions 1. To explore the presence of virulence genes (fimA, neuC and iutA) among E. coli isolates recovered from RTIs? 2. To compare the prevalence of virulence genes between upper RTIs (URTIs) and lower RTIs (LRTIs)? 3. To determine the AMR pattern among E. coli isolates?

2.4. Virulence genes identification by PCR method 2. Materials and methods Virulence genes of fimA, neuC and iutA were identified by PCR. Table 1 shows the primer sequence and specific size of the genes. Materials used for PCR included primers, dNTPs, Tag polymerase, MgCl2 and deionized water. The master mix was separately prepared for single-PCR of fimA, neuC and iutA genes. Forty-five PCR cycles were run for each gene and respective PCR conditions (Table 2) were maintained. PCR products were analysed by separation on a 1.2% (w/v) agarose gel (Johansson, 1972). The stained gel containing PCR products was observed with a UV trans illuminator (Gel Doc, Bio-Rad, USA). PCR bands were visualised with “Quality one®” software (Bio-Rad, USA) and

2.1. Sample collection The microbiology laboratory of Popular Diagnostic Centre Ltd. Dhaka 1205, Bangladesh collected sputum samples of 120 RTIs patients of whom 60 had LRTIs and 60 had URTIs between April 2017 to May 2017. Of 120 RTIs samples, 50 (41.6%) were confirmed with E. coli isolates. The 28 (56%) E. coli isolates were from URTIs and 22 (44%) were from LRTIs. We were supplied fifty E. coli isolates by the Popular Diagnostic Centre Ltd. and these isolates were transferred into Table 1 Primers used to detect virulence genes in E. coli isolated from RTIs. Gene

Primer (forward)

Primer (reverse)

Length (bp)

References

fimA neuC iutA

5′-AGTTAGGAC AGGTTCGTACCGCAT-3′ 5′-AGGTGAAAAGCCTGGTAGTGTG- 3′ 5′-CACCATGATGATAAG CAAAAAG-3′

5′-AAATAACGC GCCTGGAACGGAATG-3′ 5′-GGTGGTACATTCCGGGATGTC-3′ 5′-GACCAAAGGTGGGCC CCTGCC-3′

315 bp 675 bp 2100 bp

Hernandes et al., 2011 Zapata et al., 1992 Tokano et al., 2008

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Table 2 Cycling conditions for PCR of virulence genes. Gene

Initial denaturation

Denaturation

Annealing

Extension

Final extension

fimA neuC iutA

94 °C for 5 min 94 °C for 1 min 94 °C for 1 min

94 °C for 10 s 94 °C for 5 min 94 °C for 5 min

60 °C for 20 s 55 °C for 2 min 55 °C for 2 min

72 °C for 30 s 72 °C for 1 min 72 °C for 1 min

72 °C for 10 min 72 °C for 7 min 72 °C for 7 min

Table 3 Frequency distribution of fimA, neuC and iutA genes among E. coli isolates. Virulence gene

Positive n (%)

Negative n (%)

Total E. coli isolates (N)

fimA neuC iutA

22 (44%) 12 (24%) 0

28 (56%) 38 (76%) 0

50 50 50

Table 5 Antimicrobial resistance pattern among E. coli isolates. Antibiotics

Ampicillin Amoxicillin Cefixime Ceftriaxone Cefepime Sulfamethoxazole/ trimethoprim Ciprofloxacin Nalidixic acid Amikacin Gentamycin Colistin Imipenem Meropenem

photographed. 3. Results 3.1. Presence of fimA, neuC and iutA genes Table 3 shows the prevalence of analysed virulence genes among E. coli isolates. The frequency of fimA and neuC genes in total E. coli isolates was 44% and 24% respectively. In URTIs, the frequency distribution of fimA and neuC genes was 42.8% (12/28) and 25% (7/28) respectively. Whereas, in LRTIs, the prevalence of these genes was 45.4% (10/22) and 22.7% (5/22) respectively (Table 3). Although the prevalence of both fimA and neuC genes was more in LRTIs than URTIs, the difference did not achieve statistical significance (fimA, p = 0.854; neuC, p = 0.851) (Table 4). This resulted in null relationship between the presence of identified virulence genes and types of RTIs. We found no presence of iutA among E. coli isolates recovered either from URTIs or LRTIs.

Resistant E. coli (N = 50)

fimA (+) E. coli (N = 22)

neuC (+) E. coli (N = 12)

n

%

n

%

n

%

33 36 28 25 14 27

66 72 56 50 28 54

14 12 8 6 3 6

63.6 54.5 36.3 27.2 13.6 27.2

6 8 4 4 1 4

50 66.6 33.3 33.3 8.3 33.3

24 16 14 12 0 0 0

48 32 28 24 0 0 0

10 8 6 7 0 0 0

45.4 36.3 27.2 31.8 0 0 0

5 4 2 2 0 0 0

41.6 33.3 16.6 16.6 0 0 0

to third generation antibiotics. Approximately one-third of fimA positive and neuC positive E. coli were resistant to cefixime. No E. coli either fimA or neuC positive was resistant to colistin, imipenem and meropenem antibiotics (Table 5). More than a quarter of fimA positive E. coli showed resistance against ceftriaxone whereas, ceftriaxone resistance was comparatively higher among neuC positive E. coli (33.3%). Although low but cefepime resistance was also observed in < 15% and 10% of E. coli carrying fimA and neuC gene respectively. Resistance to aminoglycosides such as amikacin and gentamycin was found among 27.2% and 31.8% E. coli bearing fimA gene. In contrast, neuC positive E. coli showed lower resistance to amikacin and gentamycin each with 16.6%.

3.2. AMR and sensitivity pattern of E. coli AMR results showed that 100% of E. coli isolates were resistant to at least 3 antimicrobial agents tested. Therefore, all E. coli isolates were MDR. Of 50 E. coli isolates, highest sensitivity (100%) was observed to colistin, imipenem and meropenem antibiotics followed by aminoglycoside antibiotics amikacin and gentamycin (72–76%). More than half of E. coli isolates were sensitive to fluoroquinolone (52–68%), sulfa drug (56%), cephalosporin (54–72%) and penicillin (28–34%) group of antibiotics (Table 5).

4. Discussion We explored RTI E. coli isolates with regards to AMR and frequency of virulence-associated genes. The prevalence of MDR E. coli in RTIs was 100%. This is in line with the global emergence and spread of E. coli strains to cause MDR to antimicrobials. Our prevalence of MDR E. coli is comparable with the data on uncomplicated (75–95%) UTIs but much higher than complicated (40–50%) UTIs (Tan and Chlebicki, 2016). The MDR E. coli is rising because of organism's potential to mutate, acquire and transmit mobile genetic elements encoding resistant genes and harbouring virulence genes that encode virulence factors such as toxins and iron acquisition systems (Chroma and Kolar, 2010). This study found the rising prevalence of virulence genes of fimA (44%) among E. coli isolates in RTIs (Table 3). The fimA was carried by E. coli isolates for both URTIs (42.8%) and LRTIs (45.4%). However, there was no statistically significant difference (p = 0.854) in the

3.3. AMR pattern of E. coli harbouring fimA and neuC genes We investigated the AMR pattern of E. coli isolates harbouring fimA and neuC genes and results are described in Table 5. Among E. coli isolates harbouring fimA, highest resistance was found against amoxicillin (> 50% E. coli) and ampicillin (> 60% E. coli). Similarly, neuC positive E. coli showed amoxicillin resistance among two-third and ampicillin resistance among half of the isolates respectively. The second-highest resistance was found against ciprofloxacin in more than one-third of E. coli carrying fimA (45.4%) and neuC (41.6%) genes. E. coli isolates harbouring these virulence genes demonstrated resistance Table 4 Frequency distribution of fimA and neuC genes in patients with LRTIs and URTIs. Virulence gene

E. coli isolates (N = 28) in URTIs n (%)

E. coli isolates (N = 22) in LRTIs n (%)

p value at 95% CI

fimA neuC

12 (42.8%) 7 (25%)

10 (45.4%) 5 (22.7%)

0.854 0.851

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is indeed important. E. coli isolates which possessed fimA or neuC showed a quite similar antimicrobial non-susceptibility pattern. The 100% susceptibility of our E. coli isolates to colistin, imipenem, meropenem was comparable with a recent study showing > 98% susceptibility in Bangladesh. (Hasan et al., 2019). The E. coli resistance to fourth-generation cephalosporin antibiotic, cefepime (28%) was concerning. Even another study in Bangladesh showed much higher resistance of E. coli to cefepime (58%) (Hasan et al., 2019). The thirdgeneration cephalosporin resistance of E. coli (50–56%) in our study was lower than the data of South Asian countries like in Pakistan (83–89%) and in India (75–79%) (CDDEP, 2017). We found no resistance of E. coli to carbapenem antibiotics, though it was rising in Pakistan (8–12%) and India (16–20%) (CDDEP, 2017). The fluoroquinolone resistance in 32–48% E. coli was much lower than India (82–86%) and Pakistan (56–62%) (CDDEP, 2017). Although, our study had limited samples, rising of AMR in E. coli cannot be overlooked. In contrast, E. coli resistance was considerably lower in the context of the USA, UK, and Australia (CDDEP, 2017). In Bangladesh, the resistance of E. coli to aminoglycosides, third generation and fourth generation cephalosporin antibiotics was concerning. Excessive use of cephalosporin antibiotics in RTIs patients in the country may be one of the determinants of rising E. coli resistant strains. Furthermore, the preference of cephalosporin antibiotics in the hospital to avoid patients' clinical risk of using low spectrum antibiotics was common (Rahman and Huda, 2014). The longer duration and multiple courses of antibiotics were also associated with growing AMR (Costelloe et al., 2010). Evidence existed for E. coli harbouring virulence genes in extraintestinal infections such as UTIs and meningitis but not in the context of RTIs (Sarowska et al., 2019). This study demonstrated the presence of fimA and neuC in RTIs E. coli in Bangladesh. These results inform further study to confirm whether these virulence genes are the cause of MDR E. coli with a larger sample. Future efforts may reveal the distinction between the colonization of E. coli harbouring either fimA or neuC or both and the development of RTIs in the context of Bangladesh. Pathogenic E. coli have numerous virulence factors in relation to their pathogenicity, colonization and biofilm formation activities (Kuhnert et al., 2000). Therefore, mechanisms underlying the transmission of found virulence genes and the dynamics of transmission in the context of RTIs could also be worthy of investigation. To the best of our knowledge, this is the first report on the virulence genes and MDR among E. coli isolates recovered from RTIs in Bangladesh. This study has some limitations. Firstly, E. coli isolates were not characterised and classified by specific strain. Therefore, the prevalence of virulence genes was not strain specific. Secondly, the limited sample size of E. coli isolates prevented the generalization of virulence genes prevalence. This study was unable to confirm the association between AMR and virulence genes possessed by E. coli but proposes for the future.

prevalence of fimA between URTIs and LRTIs (Table 4). GuzmanHernandez et al. (2016) reported 29% prevalence of fimAagn43 gene in uropathogenic E. coli strains in Mexico. Compared to our findings, the higher prevalence of fimA was observed for E. coli K1+ (83.6%) and E. coli K1− (86.6%) strains isolated from faecal samples of pregnant women and neonates (Kaczmarek et al., 2012). These E. coli strains increase the severity of sepsis and meningitis infections in infants and mother (Kaczmarek et al., 2012). The low prevalence of fimA in our study might be explained by the sequence variability of this gene as the forward primer sequence matches a variable region of fimA (Marc and Dho-Moulin, 1996). Therefore, in future investigation, repeating the fimA PCR approach with primers in conserved regions should be employed to confirm the precise estimates of prevalence results. As fimA plays an important role to adhere to the host mucosa at different phases of infection (Johnson, 1991), the role of fimA during RTIs can further be investigated. A study showed that fimA genes can be present both in pathogenic and non-pathogenic E. coli isolates (Kuhnert et al., 2000). Therefore, it is difficult to target fimA for virulence assessment. However, we cannot avoid the influence of fimA on the pathogenicity and resistance activities of E. coli because the combination of virulence determinants helps to transform E. coli into pathogenic one (Boerlin et al., 1999; Casadevall and Pirofski, 1999). Furthermore, the research regarding the role of combination virulence determinants including fimA on AMR and E. coli pathogenicity is relatively scarce. The prevalence of neuC (24%) in RTI E. coli was much varied with literature on other infections. Our prevalence was much lower than neonatal meningitis E. coli (70%) (Wijetunge et al., 2015). However, vaginal and rectal E. coli isolates carried neuC genes with a very low propensity with 2% (n = 50) and 8% (n = 50) respectively. Watt et al. (2003) found 44% of vaginal E. coli with neuC gene. The neuC has been reported to play roles in adhesion and colonization of the avian pathogenic E. coli in chicken during RTIs (Antao et al., 2008). The neuC encodes capsule polysaccharides that protect bacteria from phagocytic activities of host and killing by complement-mediated serum (Jann and Jann, 1992; Whitfield et al., 1994). Although the roles of neuC are evidenced in uropathogenic and neonatal meningitis E. coli, future investigations are required in the context of human RTIs given our study found the presence of neuC. In our study, we found no presence of iutA in RTIs. In healthy pregnant women, the presence of iutA encoding aerobactin was reported with 28% in vaginal isolates and 34% in rectal isolates (Kaczmarek et al., 2018). A study (Hagan and Mobley, 2007) suggested that E. coli, during UTIs use iron uptake systems (iutA) that helps in intensive replication. Given, the expression of most virulence determinants on mobile genetic elements, RTIs E. coli strains might rise with different combinations of virulence genes in years ahead. This may complicate to establishing an association between literature reported virulence genes with certain categories of E. coli. Investigation on the virulence patterns of E. coli isolates in RTIs comprising of major virulence genes sets (known yet) could have great importance for their better characterisation. This exploration might help to understand the pathogenic potential of RTIs E. coli in future. A direct link between virulence traits and AMR in E. coli is evidenced (Johnson et al., 2004). Johnson et al. (2004) reported that 76 MDR E. coli isolates were resistant to ≥3 antimicrobial agents. In our study, 100% of E. coli isolates were characterised by MDR what is alarming in the context of Bangladesh. Kaczmarek et al. (2018) demonstrated a weak association between increasing virulence and increasing resistance to antibiotics when E. coli isolates were collected from vaginal and rectal samples from healthy pregnant women. Given the presence of fimA and neuC in E. coli causing RTIs, our study strongly recommends to draw similar investigation in RTIs to deeper understand the molecular mechanism of drug resistance. To guide the course of infections, assessing antibiotic susceptibility

5. Conclusions E. coli isolates recovered from RTIs harbour the virulence determinant genes of fimA and neuC in this first small-scale study. The observed low prevalence of fimA gene might be explained by the sequence variability of this gene as the forward primer sequence matches a variable region of fimA (Marc and Dho-Moulin, 1996). Reduced susceptibility of E. coli to most of the antibiotics in RTIs in Bangladesh is alarming. Further research should confirm the association between MDR E. coli and investigated virulence determinants with a larger sample from RTIs to explain the pathogenesis of E. coli in RTIs, mechanism of E. coli attaining MDR and guide optimal antibiotic therapy in future.

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Declaration of competing interest

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