Campylobacteriosis Agents in Meat Carcasses Collected from Two District Municipalities in the Eastern Cape Province, South Africa

Raw meats are sometimes contaminated with Campylobacter species from animal faeces, and meats have repeatedly been implicated in foodborne infections. This study evaluated the prevalence, virulence genes, antimicrobial susceptibility patterns, and resistance gene determinants in Campylobacter species isolated from retailed meat carcasses. A total of 248 raw meat samples were collected from butcheries, supermarkets, and open markets; processed for enrichment in Bolton broth; and incubated at 42 °C for 48 h in 10% CO2. Thereafter, the broths were streaked on modified charcoal cefoperazone deoxycholate agar (mCCDA) plates and incubated at the same conditions and for the same amount of time. After incubation, colonies were isolated and confirmed by Polymerase chain reaction using specific oligonucleotide sequences used for the identification of the genus Campylobacter, species, and their virulence markers. The patterns of antimicrobial resistance profiles of the identified isolates were studied by disk diffusion method against 12 antibiotics, and relevant resistance genes were assessed by PCR. From culture, 845 presumptive Campylobacter isolates were obtained, of which 240 (28.4%) were identified as genus Campylobacter. These were then characterised into four species, of which C. coli had the highest prevalence rate (22.08%), followed by C. jejuni (16.66%) and C. fetus (3.73%). The virulence genes detected included iam (43.14%), cadF (37.25%), cdtB (23.53%), flgR (18.63%), and flaA (1.96%), and some of the isolates co-harboured two to four virulence genes. Of the 12 antibiotics tested, the highest phenotypic resistance displayed by Campylobacter isolates was against clindamycin (100%), and the lowest level of resistance was observed against imipenem (23.33%). The frequency of resistance genes detected included catll (91.78%), tetA (68.82%), gyra (61.76%), ampC (55%), aac(3)-IIa (aacC2)a (40.98%), tetM (38.71%), ermB (18.29%), tetB (12.90%), and tetK (2.15%). There is a high incidence of Campylobacter species in meat carcasses, suggesting these to be a reservoir of campylobacteriosis agents in this community, and as such, consumption of undercooked meats in this community is a potential health risk to consumers.


Introduction
In the last decade, there has been a global upsurge in the rate of Campylobacter infections [1,2], and Campylobacter has emerged as one of the most significant bacteria of public health importance [3]. Globally, Campylobacter infection is a significant zoonosis, considered to be the leading cause of bacterial foodborne infection [4]. This zoonotic infection is of great public health concern [5], with meats known as the major risk factor [6] due to consumption of undercooked poultry or red meats [7]. Worldwide,

Sample Collection
A total number of 258 meat samples (mutton, chicken, turkey, beef, and pork) were purchased from different retail markets, open markets, and butcheries in different locations and towns in Chris Hani and Amathole District Municipalities, Eastern Cape, South Africa. All the meat samples were aseptically packed into separate sterile plastic bags to prevent cross contamination and were transported to the laboratory for analysis in a cooler box with ice packs within six hours of collection.

Microbiological Analysis of the Meat Samples
The meat samples were analysed following ISO 10,272 guidelines for isolation and identification of Campylobacter species [37,38]. Briefly, 25 g portions of the meat samples were homogenised in 245 mL of buffered peptone water (M614-500G, (Vadhani, Mumbai, India)). Thereafter, 10 mL of the homogenate was added into 90 mL of Bolton selective enrichment broth (1.00068.0500 Merck), to which Bolton broth selective supplement (1.00079.0010 Merck) with 5% (v/v) defibrinated horse blood (JMS, Singapore) was added, and the resulting mixture was incubated at 42 °C for 48 h under microaerophilic conditions in 10% CO2 in an HF151UV CO2 incubator. After the 48 h incubation period, a loopful of the inoculum from the enriched broth was streaked on modified charcoal cefoperazone deoxycholate agar (mCCDA) plates containing antibiotic selective supplement (CCDA selective supplement 1.00071.0010) and incubated under the same conditions and for the same amount of time stated above. Thereafter, colonies suspected to be Campylobacter based on colony morphology were picked and re-streaked onto blood agar base plates supplemented with 7% (v/v) defibrinated horse blood, and the plates were incubated under the same conditions and for the same amount of time.

DNA Extraction
Template DNA for PCR assay was extracted following the process described by Sierra-Arguello et al. [39] with slight modification. Briefly, colonies isolated from the blood agar plates were grown in 5 mL of Tryptone Soya Broth (TSB) for 48 h at 42 °C under microaerobic conditions in a 10% CO2 incubator. After incubation, 1 mL of the broth was centrifuged at 12,800 rpm for 5 min, and the cells were suspended in 400 µL of sterile distilled water in sterile 1.5 mL Eppendorf tubes. The suspensions were boiled for 10 min at 100 °C in a heating block and allowed to cool, after which the suspensions

Sample Collection
A total number of 258 meat samples (mutton, chicken, turkey, beef, and pork) were purchased from different retail markets, open markets, and butcheries in different locations and towns in Chris Hani and Amathole District Municipalities, Eastern Cape, South Africa. All the meat samples were aseptically packed into separate sterile plastic bags to prevent cross contamination and were transported to the laboratory for analysis in a cooler box with ice packs within six hours of collection.

Microbiological Analysis of the Meat Samples
The meat samples were analysed following ISO 10,272 guidelines for isolation and identification of Campylobacter species [37,38]. Briefly, 25 g portions of the meat samples were homogenised in 245 mL of buffered peptone water (M614-500G, (Vadhani, Mumbai, India)). Thereafter, 10 mL of the homogenate was added into 90 mL of Bolton selective enrichment broth (1.00068.0500 Merck), to which Bolton broth selective supplement (1.00079.0010 Merck) with 5% (v/v) defibrinated horse blood (JMS, Singapore) was added, and the resulting mixture was incubated at 42 • C for 48 h under microaerophilic conditions in 10% CO 2 in an HF151UV CO 2 incubator. After the 48 h incubation period, a loopful of the inoculum from the enriched broth was streaked on modified charcoal cefoperazone deoxycholate agar (mCCDA) plates containing antibiotic selective supplement (CCDA selective supplement 1.00071.0010) and incubated under the same conditions and for the same amount of time stated above. Thereafter, colonies suspected to be Campylobacter based on colony morphology were picked and re-streaked onto blood agar base plates supplemented with 7% (v/v) defibrinated horse blood, and the plates were incubated under the same conditions and for the same amount of time.

DNA Extraction
Template DNA for PCR assay was extracted following the process described by Sierra-Arguello et al. [39] with slight modification. Briefly, colonies isolated from the blood agar plates were grown in 5 mL of Tryptone Soya Broth (TSB) for 48 h at 42 • C under microaerobic conditions in a 10% CO 2 incubator. After incubation, 1 mL of the broth was centrifuged at 12,800 rpm for 5 min, and the cells were suspended in 400 µL of sterile distilled water in sterile 1.5 mL Eppendorf tubes. The suspensions were boiled for 10 min at 100 • C in a heating block and allowed to cool, after which the suspensions were centrifuged at 12,800 rpm for 5 min and the supernatants were collected and stored at −20 • C until ready for use.

Molecular Identification of the Genus Campylobacter
A 439 bp part of the 16S rRNA gene was amplified using primer CAM220 F-GGTGTAGGATGAGACTATATA and CAM659 R-TTCCATCTGCCTCTCCC as reported by Moreno et al. [40]. A singleplex PCR assay was carried out in a 25 µL reaction volume (5 µL of the DNA, 12.5 µL master mix, 2 µL of primer, and 5.5 µL of nuclear free water), and the PCR cycling conditions were set at initial denaturation (95 • C for 5 min), followed by 33-cycle (94 • C for 1 min, 58 • C for 1 min, and 72 • C for 2 min), and the final extension was set at 72 • C for 2 min. Verification of the amplified PCR products was carried out by resolving them in 1.5% agarose gel stained with ethidium bromide at 135 volts for 30 min, which was detected under a short-wavelength UV light source; C. jejuni ATCC 33.560 was used as the positive control.

Molecular Classification of Campylobacter Species
PCR amplification was further carried out to delineate the isolates to the species level for the detection of C. lari, C. fetus, C. jejuni, and C. coli. The primer sets used for the detection of these species are as reported by Yamazaki-Matsune et al. [41].

Molecular Detection of Virulence Genes
The identified Campylobacter species were further screened for the presence of invasion genes (ciaB and iam), adherence genes (flaA and cadF), a toxin gene (cdtB) and flagella synthesis, and a modification gene (flgR). The primer sets for the detection of cdtB, flaA and cadF genes were used as reported by Modi et al. [24], iam gene [42], ciaB gene [43] and flgR) gene [44].

Multiple Antibiotic Resistance (MAR) Index
For the determination of the multiple antibiotic resistance (MAR) index, the formula MAR = x/y, stated by Krumperman [46], was adopted where x = is the number of antibiotics to which the test isolate showed resistance and y = is the total number of antibiotics to which the test isolate has been evaluated for susceptibility.

Genotypic Assessment of Antibiotic Resistance Genes
Molecular screening of resistance genes was carried out on important resistance genes by both simplex and multiplex PCR assays on the isolates, which showed phenotypic resistance to the test antibiotics. The primer sets used for the detection of tetA, tetB, tetC and tetD genes were used as reported by Ng et al. [47], tetK and tetM genes [48], gyrA gene [49], ermB gene [50], catI and catII genes [51], (aac(3)-IIa (aacC2) a gene [52] and IMI, KPC, VIM and bla OXA -48-lik genes [53]. Verification of the amplified PCR products was carried out as stated above.

Molecular confirmation of Campylobacter species
In the effort to isolate and detect Campylobacter species in meat samples, including pork, mutton, mutton offals, beef, beef offals, turkey, chicken, and chicken offals; the samples were subjected to both traditional culture and PCR techniques. From culture, a total of 845 presumptive isolates were obtained, of which 28.40% (208/845) were identified as belonging to the genus Campylobacter by PCR assay, of which 32.5% (208/640) were obtained from retail markets, 15.17% (22/145) from butcheries, and 16.67% (10/60) from open markets. The detailed results of the number of isolates from various meat types are shown in Table 1, while Figure 2 shows a representative gel image of the PCR confirmed genus Campylobacter.

Genotypic Assessment of Antibiotic Resistance Genes
Molecular screening of resistance genes was carried out on important resistance genes by both simplex and multiplex PCR assays on the isolates, which showed phenotypic resistance to the test antibiotics. The primer sets used for the detection of tetA, tetB, tetC and tetD genes were used as reported by Ng et al. [47], tetK and tetM genes [48], gyrA gene [49], ermB gene [50], catI and catII genes [51], (aac(3)-IIa (aacC2) a gene [52] and IMI, KPC, VIM and blaOXA-48-lik genes [53]. Verification of the amplified PCR products was carried out as stated above.

Molecular confirmation of Campylobacter species
In the effort to isolate and detect Campylobacter species in meat samples, including pork, mutton, mutton offals, beef, beef offals, turkey, chicken, and chicken offals; the samples were subjected to both traditional culture and PCR techniques. From culture, a total of 845 presumptive isolates were obtained, of which 28.40% (208/845) were identified as belonging to the genus Campylobacter by PCR assay, of which 32.5% (208/640) were obtained from retail markets, 15.17% (22/145) from butcheries, and 16.67% (10/60) from open markets. The detailed results of the number of isolates from various meat types are shown in Table 1, while Figure 2 shows a representative gel image of the PCR confirmed genus Campylobacter.

Molecular Characterisation of Campylobacter Species
The 240 isolates identified as belonging to the genus Campylobacter were further delineated into four Campylobacter species by PCR technique: 53 (22.08%) isolates were identified as C. coli, 40 (16.66%) as C. jejuni, and 9 (3.75%) as C. fetus, whereas C. lari was not detected. A summary of the numbers of Campylobacter species identified in the meat types is shown in Table 2

Molecular Characterisation of Campylobacter Species
The 240 isolates identified as belonging to the genus Campylobacter were further delineated into four Campylobacter species by PCR technique: 53 (22.08%) isolates were identified as C. coli, 40 (16.66%) as C. jejuni, and 9 (3.75%) as C. fetus, whereas C. lari was not detected. A summary of the numbers of Campylobacter species identified in the meat types is shown in Table 2, while Figures 3 and 4 are representative gel electrophoresis images of PCR-confirmed C. jejuni, C. coli, and C. fetus, respectively.
Percentage distribution pattern of detected virulence genes in the identified Campylobacter species.

Assessment of Resistance Determinants
The resistance genes screened among the phenotypic resistant-Campylobacter isolates are listed in Table S3, and the choice for the selection of these genes was centred on their high phenotypic resistance rates. Thus, 12 antibiotic resistance genes were screened for probable detection of resistance genes among the identified Campylobacter species and also to determine the pattern of occurrence of multiple resistance genes in the isolates. From the PCR results obtained, the order of the frequency level of the resistance genes detected was as follows: catll (91.78%), tetA (68.82%), gyra (61.76%), ampC (55%), aac(3)-IIa (aacC2) a (40.98%), tetM (38.71%), ermB (18.29%), tetB (12.90%), and tetK (2.15%). In contrast, the IMI, KPC, VIM, bla OXA -48-like, catl, tetC, tetD, and tetK genes were not detected. Figures 8 and 9 are representative gel images of the amplified PCR products of the assessed antibiotic resistance genes. Similarly, the patterns of the level of detected multiple resistance genes are shown in Table 6.

Assessment of Resistance Determinants
The resistance genes screened among the phenotypic resistant-Campylobacter isolates are listed in Table S3, and the choice for the selection of these genes was centred on their high phenotypic resistance rates. Thus, 12 antibiotic resistance genes were screened for probable detection of resistance genes among the identified Campylobacter species and also to determine the pattern of occurrence of multiple resistance genes in the isolates. From the PCR results obtained, the order of the frequency level of the resistance genes detected was as follows: catll (91.78%), tetA (68.82%), gyra (61.76%), ampC (55%), aac(3)-IIa (aacC2) a (40.98%), tetM (38.71%), ermB (18.29%), tetB (12.90%), and tetK (2.15%). In contrast, the IMI, KPC, VIM, blaOXA-48-like, catl, tetC, tetD, and tetK genes were not detected. Figures  8 and 9 are representative gel images of the amplified PCR products of the assessed antibiotic resistance genes. Similarly, the patterns of the level of detected multiple resistance genes are shown in Table 6.

Discussion
Reports on the prevalence, virulence marker, and antimicrobial resistance genes in Campylobacter isolates recovered from retailed meat samples are well documented in some parts of the world, but limited information is available in some provinces in South Africa, particularly in the Eastern Cape Province. Hence, this study aimed to address the prevalence and characterise the identified Campylobacter species, virulence genes, and resistance genes in Campylobacter isolates recovered from meat carcasses. Campylobacter species are major bacteria foodborne enteropathogens that are regularly spread to humans through the consumption of contaminated food including meats [54]. In this study, a high rate of Campylobacter was detected in 240 (28.40%) isolates from meat carcasses, and this gives valuable insight into the possible risks of foodborne infection to humans. Studies carried out in Italy by Stella et al. [55], in Malaysia [56], in China [57], in France [58], and in South Korea [59] also detected Campylobacter in meat samples with detection rates of 34.10%, 50.9%, 48.9%, 76%, and 31.67%, respectively, and our findings are in line with their reports. Other studies carried out in Spain by García-Sánchez et al. [60], in Pakistan by Nisar et al. [3], in Yangzhou, China by Zou et al. [61], and in Northern Poland by Andrzejewska et al. [62] also detected Campylobacter in meat samples, and these results are also akin with their reports. According to Seliwiorstow et al. [63], different sampling sources make an impact on the occurrence rates of Campylobacter, which indicate the risk factors associated with handling. The occurrence rates of Campylobacter detected in the different market sources are in the order of 32.5% (retail markets), 16.67% (open markets), and 15.17% (butcheries). The highest occurrence rates of the genus Campylobacter were also detected in isolates from mutton (44.4%), followed by beef (34%), turkey (31.3%), beef offals (31%), chicken offals (29%), chicken (27%), and pork (25.2%). The occurrence rate of Campylobacter in mutton samples was much higher than that in other meat types, and this result is in agreement with the report of Maktabi et al. [64].
In the present study, the high detection rate of Campylobacter was observed in isolates from beef carcasses, and this result also corroborates the reports of Kashoma et al. [65] and Premarathne et al. [66]. The high rates of Campylobacter detection in turkey samples observed in this study also support those reported by Noormohamed and Fakhr [67] in Oklahoma, USA (17%), and Szosland-Fałtyn et al. in Poland (47.37%) [68]. The 240 isolates identified belonging to the genus Campylobacter were then characterised into four species, of which 53 (22.08%) were identified as C. coli, 40 (16.66%) as C. jejuni, and 9 (3.73%) as C. fetus, whereas C. lari was not detected. Higher rates of C. coli and C. jejuni were detected than other Campylobacter species, and our findings corroborate the report of Hodges et al. [69], Ocejo et al. [70], Sulaiman et al. [71], and Vinueza-Burgos et al. [72]. In contrast, a low prevalence rate of C. fetus was detected, and this finding is also in agreement with the report of Sinulingga et al. [56]. C. coli and C. jejuni are known to cause infection in humans, but from the first report of Campylobacter infection to date, Campylobacter pathogenesis has not been clearly understood. Though, what has been clear about Campylobacter infections and has been proposed as virulence determinants includes Campylobacter's invasive capability, adherence to intestinal mucosa, ability to produce toxins, and flagella-mediated motility [73]. Thus, the presence of these specific genes associated with Campylobacter invasion, adhesion, toxin production, and colonisation are all essential for the process of infection, and the mechanism by which they cause disease in humans is assumed to be multifactorial [74]. From the PCR results of the virulence gene assessed, most Campylobacter species were detected to harbour a high proportion of cadF, flaA, and iam genes responsible for colonisation, invasion, and adherence, and our results corroborate the report of Abu-Madi et al. [75]. The iam gene had the highest occurrence rate of 43.14% (Table 3) among the various virulence genes screened, and our results also corroborate the report of Redondo et al. [76].
Similarly, the high occurrence rate of the cadF (37.25%) gene and the lower prevalence of the flaA (1.96%) gene were detected in the Campylobacter isolates from meat samples, and these results are akin with the reports of Andrzejewska et al. [77] and Ripabelli et al. [78]. Our results also corroborated the report of Chukwu et al. [74], who also detected the cadF gene in Campylobacter strains isolated from water and paediatric stools. The ciaB gene was screened for but was not detected, and our findings are contrary to the reports of Melo et al. [79], Melo et al. [80], and Zhong et al. [81] who reported high detection rates of the ciaB gene in meats and in retailed food samples. Another virulence gene screened for was the cdtB gene, and the presence and expression of any of the cdt genes (cdtA, cdtB, and cdtC) are essential for the efficient activity of the CDT toxin. In this study, the cdtB gene was observed to be widespread among the Campylobacter species, with C. coli strains revealed to have a higher prevalence rate of cdtB than C. jejuni and C. fetus (Table 3), and our results are not in agreement with the reports of Wieczorek et al. [82] and Reddy and Zishiri [83], which reported a higher prevalence rate of the cdtB gene in C. jejuni than in the C. coli strain. Multiple virulence genes were also detected in both C. coli and C. jejuni with C. coli observed to harbour more virulence genes than C. jejuni, and these results are in contrast with the reports of Lim et al. [84] and Zhang et al. [85]. Campylobacter isolates have also been reported by Han et al. [86] to co-harbour more than three virulence genes, and our findings corroborate this report. Virulence genes in the Campylobacter genome are known to be implicated in human infection, and Campylobacter pathogenicity may be strengthened by the expression of a single virulence gene or multiple virulence genes that are enough to establish infection in the host [80].
The dissemination of virulence-associated genes in the identified Campylobacter strains isolated from meats showed a potential risk to humans and an impending risk of outbreak of Campylobacter infection if appropriate measures are not put into place. From the antimicrobial susceptibility testing result, a total of 78 antibiotic resistance profiles were generated among the 102 Campylobacter isolates characterised as C. coli, C. jejuni, and C. fetus, and 76.47% were resistant to more than two antimicrobial families and were classified as multi-drugs resistant strains ( Table 5). The most common observed resistant profile was LEV-CRO-C-CIP-E-ATH-IMI-CD-T-GM-DXT-AP, which was common among C. coli and C. jejuni strains. The isolates displayed high phenotypic resistance to tetracycline (94.17%), erythromycin (87.03%), ampicillin (97.08%), and ciprofloxacin (76.25%). In Brazil, studies have shown that there is a high prevalence of quinolones (72.2%), tetracycline (43%), erythromycin (38.9%), and ampicillin (26.9%) resistance in Campylobacter in circulation [87,88]. In Bolivia, Argentina, Chile, and Peru, many Campylobacter strains are resistant to quinolones (47-78%) as well as tetracycline (40.8-65.9% in Argentina and Bolivia), erythromycin (58.6% to 61.4% in Bolivia and Chile), and ampicillin (47.2% in Argentina) [89]. The value of the MAR index is 0.2, and the MAR index is a good risk assessment tool used to distinguish high-and low-risk areas where antibiotics are overused [90]. The MAR indices of the isolates were calculated, and 77.83% were found to have a MAR index greater than 0.2, while 17 isolates had MAR indices of 1.0 (Table 5). A MAR index value of greater than 0.2 indicates a high-risk source of contamination where antibiotics are often used, and based on our results, most isolates had MAR indices of greater than 0.2, confirming that there is high selective pressure and high antibiotic use in these areas.
Because the effectiveness of antibiotic resistance might be compromised in the treatment of infections, antimicrobial resistance genes were screened. Multiple antibiotic resistance genes were highly detected in most of the isolates (Table 6), and this is in agreement with the multiple phenotypic resistance profiles displayed by the isolates (Table 5). Multiple resistance genes in the Campylobacter isolates were detected, and our results are in agreement with the reports of Chukwu et al. [74] and Wieczorek et al. [91]. There was a high rate of detection of resistance genes in chloramphenicol (catll (91.78%)), tetracycline (tetA (68.82%)), ciprofloxacin (gyra (61.76%)), ampicillin (ampC (55%)), gentamycin (aac(3)-IIa (aacC2) a (40.98%)), and tetracycline (tetM (38.71%) in the Campylobacter isolates. In this study, the high detection rate of resistance genes in tetracycline/ciprofloxacin-Campylobacter isolates was responsible for its phenotypic resistance, and this is in line with the report of Nguyen et al. [92]. A high rate of the gyra gene was also detected in the Campylobacter isolates recovered from meat carcasses, and our results are also in agreement with the report of Du et al. [93]. The results from the study of Rahimi et al. [94] show that Campylobacter isolates recovered from meat carcasses were all susceptible to chloramphenicol and gentamycin, and our results are contrary to their report. The implications in antimicrobial resistance show a strong connection between the use of antibiotics in animal production, veterinary medicine, and antibiotic-resistant Campylobacter isolates in humans [95], although the majority of the antibiotics used in the treatment of bacterial infections in humans are also used in animals. Nevertheless, a One Health approach to addressing the issue of antibiotic resistance and the spread of antibiotic-resistant bacteria includes plans to maintain and carry on with the efficiency of current antibiotics by abolishing their inappropriate use and by preventing the spread of infection [96]. Another One Health approach in the prevention of human campylobacteriosis and the solution to the spreading of antibiotic-resistant bacteria is to improve animal, environmental, and human health with key components such as access to safe food, clean water, and hygiene [97]. In addition, recent One Health approach in the prevention and spread of antimicrobial resistance have focused primarily on the reduction of the use of antibiotics in food animals [98].

Conclusions
We investigated the prevalence rate, characterisation, distribution patterns of virulence genes, antibiotic susceptibility patterns, and antibiotic resistance genes in Campylobacter species isolated from meat carcasses obtained from butcheries, open markets, and supermarkets. High prevalence rates of the genus Campylobacter and virulence markers were detected in meat samples obtained in Chris Hani and Amathole District Municipalities, Eastern Cape, South Africa, and PCR assay is one of the appropriate methods for the detection and characterisation of virulence genes and resistance genes in bacteria species. The majority of the isolates showed resistance to the test antibiotics, and multi-resistant isolates were also observed. In conclusion, there should be a continues surveillance of the presence of these pathogens and antibiotic resistance genes in Campylobacter isolates, and awareness of the impending risks associated with the consumption of undercooked, contaminated meats should be increased.

Supplementary Materials:
The following are available online at http://www.mdpi.com/2304-8158/9/2/203/s1, Table S1: Primer sets used for the identification of different Campylobacter species; Table S2: Oligonucleotide sequences used for the detection of virulence genes; Table S3: Primer sequences used for the detection of resistance genes.
Author Contributions: A.I. carried out the experiment, analysed the results, and wrote the paper. A.I.O. applied for funding, designed the project, supervised the work, and corrected and proofread the paper. All authors have read and agreed to the published version of the manuscript.