First Report of aac(6′)-Ib and aac(6′)-Ib-cr Variant Genes Associated with Mutations in gyrA Encoded Fluoroquinolone Resistance in Avian Campylobacter coli Strains Collected in Tunisia

The aac(6′)-Ib gene is the most widespread gene encoding aminoglycoside-modifying enzyme and conferring resistance to tobramycin, streptomycin and kanamycin. The variant aac(6′)-Ib-cr gene confers resistance to both aminoglycosides and fluoroquinolones (FQ). A total of 132 Campylobacter isolates, including 91 C. jejuni and 41 C. coli, were selected from broiler hens isolates. The aac(6′)-Ib gene was amplified using PCR and was subsequently digested with the BtsCI restriction enzyme to identify aac(6′)-Ib-cr. Among these isolates, 31 out of 41 C. coli (75.6%) and 1 (0.98%) C. jejuni were positive for the aac(6′)-Ib gene, which was identified as the aac(6′)-Ib-cr variant in 10 (32.25%) C. coli isolates. This variant was correlated with mutations in gyrA (Thr-86-Ile), as well as resistance to FQs. This study is the first report in Tunisia on Campylobacter coli strains harboring both the aac(6′)-Ib and aac(6′)-Ib-cr variants. These genes were present in Campylobacter isolates exhibiting resistance to multiple antibiotics, which restricts the range of available treatments.


Introduction
Campylobacter is an important zoonotic pathogen, and campylobacteriosis constitutes a public health problem worldwide.Fluoroquinolones are one of the alternative treatments for invasive infections caused by Campylobacter in adult patients.The emergence of antimicrobial resistance involves some therapeutic failures or restrictions in the use of antimicrobial agents.However, high-level fluoroquinolone resistance is relatively uncommon in Campylobacter, probably due to the prohibitive fitness cost of the bacteria [1,2].Quinolone/fluoroquinolone resistance is mainly caused by chromosomal mutations in the genes that control the production of efflux pumps or bacterial topoisomerases (DNA gyrase and topoisomerase IV), or both [3].However, transferable mechanisms known as plasmid-mediated quinolone resistance (PMQR) have emerged during recent decades especially in Gram-negative bacteria [4].PMQR determinants are commonly reported in fluoroquinolone-resistant Enterobacteriaceae and include qnr (qnrA, qnrB, qnrC, qnrD, qnrS and qnrVC), qep, oqxAB or aac(6 )-Ib-cr genes [5].The aminoglycoside acetyltransferase AAC(6 )-Ib enzyme, which confers resistance to tobramycin, kanamycin, and amikacin, was first identified in Klebsiella pneumoniae isolates in 1986.Since then, several variants of this enzyme have been described [6,7].The aac(6 )-Ib-cr variant of aac(6 )-Ib encodes an aminoglycoside acetyltransferase that contributes reduced susceptibility to ciprofloxacin via N-acetylation of its piperazinyl amine [8].Thus, AAC(6 )-Ib-cr is a bifunctional acetyltransferase, capable of modifying both aminoglycosides and quinolones.The AAC(6 )-Ib-cr enzyme contains two amino acid modifications, Asp179Tyr and Trp102Arg [9].Globally, this enzyme is widely reported, either plasmid or chromosomally encoded, from Gram-negative bacteria, especially in Enterobacteriaceae of human, farm animal and environmental origins, showing its great potential to be disseminated [10].In general, in bacteria, the spread of genes encoding antimicrobial-resistance is mediated by mobile genetic elements such as plasmids, transposons and insertion sequences as well as integrons, and rarely by phages, which enhance their rapid and large-scale spread [11].
Fluoroquinolones are among the three most widely used antimicrobials (macrolides and tetracyclines) used to treat systemic infection caused by Campylobacter; in addition, gentamicin (an aminoglycoside) is also used in some cases [1,2].Taken together, it is crucial to identify the strains harboring the aac(6 )-Ib-cr variant in order to employ fluoroquinolone antimicrobials appropriately in human therapy.The aim of this study is to present the characteristics of campylobacter isolates from poultry origin harboring the aac(6 )-Ib-cr variant.This study, the first of its kind in Tunisia, will be useful for the future studies of emerging genes conferring antibiotic resistance.

Detection of Mutations in GyrA
Nearly 90% of C. jejuni and 80% of C. coli isolates that were resistant to ciprofloxacin and/or nalidixic acid had the Thr-86-Ile amino acid substitution in their GyrA, as determined using MAMA-PCR.The Thr-86-Ile alteration was not found in one C. jejuni isolate that was susceptible to both ciprofloxacin and/or nalidixic acid.

aac(6′)-Ib Sequencing and Phylogenetic Analysis
Among the isolates harboring the aac(6′)-Ib gene based on their antibio profiles and BtsCI digestion results, the aac(6′)-Ib of one C. jejuni isolate an isolates (one harboring the aac(6′)-Ib-cr) was sequenced.The sequences of gene were deposited in GenBank under accession numbers OR124788 OR125070, OR125069, and OR192836.
A phylogenetic analysis was performed to observe the phylogenetic rel aac(6′)-Ib gene among other bacterial species.The sequence analysis show sian aac(6′)-Ib sequences found in this study (OR124788, OR125068, OR12507 and OR192836) were closely related to other sequences from various specie terobacter cloacae, Citrobacter freundii, and Pseudomonadaceae, and the phyloge was supported by a high bootstrap value of 99% (Figure 2).Moreover, clu the maximum likelihood method defined four sequence clusters and one s responding to C. jejuni AF4339785 (aacA4) (Figure 2).Different clusters can e properties, while others show substantial differences in their characteristics.

aac(6 )-Ib Sequencing and Phylogenetic Analysis
Among the isolates harboring the aac(6 )-Ib gene based on their antibiotic resistance profiles and BtsCI digestion results, the aac(6 )-Ib of one C. jejuni isolate and four C. coli isolates (one harboring the aac(6 )-Ib-cr) was sequenced.The sequences of the aac(6 )-Ib gene were deposited in GenBank under accession numbers OR124788, OR125068, OR125070, OR125069, and OR192836.
A phylogenetic analysis was performed to observe the phylogenetic relationships of aac(6 )-Ib gene among other bacterial species.The sequence analysis showed that Tunisian aac(6 )-Ib sequences found in this study (OR124788, OR125068, OR125070, OR125069, and OR192836) were closely related to other sequences from various species, such as Enterobacter cloacae, Citrobacter freundii, and Pseudomonadaceae, and the phylogenetic branch was supported by a high bootstrap value of 99% (Figure 2).Moreover, clustering using the maximum likelihood method defined four sequence clusters and one singleton corresponding to C. jejuni AF4339785 (aacA4) (Figure 2).Different clusters can exhibit similar properties, while others show substantial differences in their characteristics.
Figure 2. Molecular phylogenetic analysis by maximum likelihood method.The evolutionary history was inferred by using the maximum likelihood method based on the Kimura two-parameter model.The tree with the highest log likelihood (−1721.35) is shown.The percentage of trees in which the associated taxa clustered together is shown next to the branches.Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach, and then selecting the topology with superior log likelihood value.A discrete Gamma distribution was used to model evolutionary rate differences among sites (five5 categories (+G, parameter = 200.0000)).The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 8.19% sites).The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.The analysis involved 34 nucleotide sequences.All positions containing gaps and missing data were eliminated.There were a total of 421 positions in the final dataset.Evolutionary analyses were conducted in MEGA7.ITOL version 6 was used to create the phylogenetic tree.The strains in our investigation are described using their bolded accession number code.

Discussion
In order to counter the selection pressure brought on by the use of antibiotics in both veterinary and human medicine, Campylobacter has developed a variety of molecular mechanisms of antibiotic resistance, such as modification or mutation of antimicrobial for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach, and then selecting the topology with superior log likelihood value.A discrete Gamma distribution was used to model evolutionary rate differences among sites (five5 categories (+G, parameter = 200.0000)).The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 8.19% sites).The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.The analysis involved 34 nucleotide sequences.All positions containing gaps and missing data were eliminated.There were a total of 421 positions in the final dataset.Evolutionary analyses were conducted in MEGA7.ITOL version 6 was used to create the phylogenetic tree.The strains in our investigation are described using their bolded accession number code.

Discussion
In order to counter the selection pressure brought on by the use of antibiotics in both veterinary and human medicine, Campylobacter has developed a variety of molecular mechanisms of antibiotic resistance, such as modification or mutation of antimicrobial targets, modification or inactivation of antibiotics, and decreased drug accumulation by drug efflux pumps.
According to our findings, the rate of ciprofloxacin resistance (99.2%) in Campylobacter isolates is quite high.The fact that the same antimicrobials are frequently used as first-line treatments in broiler flocks to combat bacterial illnesses can be blamed for the high rate of resistance to ciprofloxacin, which is equivalent to that found in Italy [12][13][14].Several studies have unequivocally demonstrated a link between the use of fuoroquinolones in chicken production and a rise in the resistance of Campylobacter isolates of avian and human origins [15].When considered together, the excessive and unreasonable use of antimicrobial medications is clearly related to the high rates of antibiotic resistance.There are two different mechanisms that contribute to ciprofloxacin resistance.The first is the presence of the cmeABC operon, the most common Campylobacter multidrug efflux pump, which contributes to fluoroquinolone resistance by reducing the amount of drug in cells [16,17].Amino acid substitutions in the quinolone resistance-determining region (QRDR) of the gyrA gene, particularly the Thr-86-Ile mutation, common in fluoroquinolone-resistant isolates, is the second mechanism of resistance [18,19].This mutation was found in 90% and 80% of quinolone-resistant C. jejuni and C. coli isolates, respectively.Since the remaining resistant isolates have the cmeB gene and do not have the Thr-86-Ile substitution, as reported in our previous work on these isolates [20], the observed resistance may be due to the action of an efflux pump or to the presence of additional alterations in the ParC subunit of topoisomerase IV.
On the other hand, we showed high rates of resistance to kanamycin (28.03%), tobramycin (58.33%), and streptomycin (48.48%), which are comparable to rates reported in the USA and China [20].In fact, high levels of aminoglycoside resistance are due to the use of these antimicrobial drugs in recent years in humans and their administration to treat occasional systemic infections caused by Campylobacter; the emergence and spread of these resistance markers represent a potential concern for public health, especially concerning gentamicin resistance, which is an antibiotic occasionally used in systemic infection caused by Campylobacter isolates [21].
Notably, new antibiotic resistance mechanisms continuously emerge in Campylobacter.Aminoglycoside phosphotransferases constitute the majority of aminoglycoside-modifying enzymes identified in Campylobacter spp.They mediate kanamycin and neomycin resistance.The AAC(6 )-Ib is a clinically significant enzyme commonly present in a number of Gramnegative bacteria.It confers resistance to tobramycin, kanamycin, and amikacin.The AAC(6 )-Ib enzyme is interesting not only because of its widespread distribution, but also because the aac(6)-Ib gene is frequently located on integrons, transposons, plasmids, genomic islands, and other genetic structures.In addition, the aac(6 )-Ib-cr mutant gene encodes resistance to both fluoroquinolones and aminoglycosides.The frequency of aac(6 )-Ib-cr found in this study exceeds those found in previous studies in Brazil (up to 4%) in clinical Pseudomonas aeruginosa and Providencia stuartii isolates [22,23] and from samples obtained in different countries, which ranged from 12% to 23.3% [24,25].Notably, the use of broad-spectrum bactericidal aminoglycosides routinely in children, primarily for infections caused by Gram-negative fluoroquinolone-resistant pathogens, has increased worldwide [26].Therefore, this use of aminoglycosides would have increased the likelihood occurrence of various transferable genes encoding resistance against them including wildtype aac(6 )-Ib.Consequently, the emergence of the aac(6 )-Ib-cr variant could be the result of an enormous number of silent mutations in this wild gene, of which one has acquired the advantage of being a bi-functional enzyme, the 'cr" variant.In addition, the worry for this variant is that it substantially enhanced the frequency of selection of chromosomal mutations in DNA gyrase and topoisomerase IV upon exposure to ciprofloxacin [27,28].
In our isolates, the aac(6 )-Ib-cr variant among those harboring aac(6 )-Ib was extremely high (31.25%),and only identified among C. coli isolates.This rate was much higher than those reported E. coli and other Enterobactereaceae isolates in other countries (4-15.6%)[29][30][31].However, among clinical E. coli isolates collected in Shanghai, China, in 2000 to 2001, 51% had the 'cr' variant of aac(6 )-Ib [30].As discussed above, all studies dealing with aac(6 )-Ib-cr were performed on isolates belonging to Gram-negative bacteria; however, to the best of our knowledge, only one study has reported the occurrence of an aacA4 allele (an aminoglycoside 6 -N-acetyltransferase) from a C. jejuni strain isolated from a broiler chicken house environment, which demonstrated 100% homology to the AAC(6 )-Ib 7 enzymes that confer characteristics resulting in higher kanamycin and tobramycin MICs, but only slightly increased resistance to gentamicin [32].Therefore, we report for the first time the occurrence of aac(6 )-Ib-cr in Campylobacter isolates.
Furthermore, clustering using a maximum likelihood method based on the Kimura 2-parameter model was performed.Different clusters can exhibit similar properties, while others show substantial differences in their characteristics such as those cases in which there are significant specificity variations.The tree with the highest log likelihood (−1721.35) is shown.The percentage of trees in which the associated taxa clustered together is shown next to the branches.Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach, and then selecting the topology with a superior log likelihood value.The analysis involved 34 nucleotide sequences.Phylogenetic analyses showed four sequence clusters and one singleton corresponding to C. jejuni AF4339785 (aacA4).The aac(6 )-Ib sequences of our five isolates (accession no: OR124788, OR125068, OR125070, OR125069, and OR192836) clustered with sequences derived from different species and countries.Using the information available, it is still not clear whether all aac(6 ) enzymes evolved from a single origin, or whether the four groups are less related and the 6 acetylating activity has evolved independently at least three times [33].According to the phylogenetic analyses reported by Salipante and Hall [33], the aac(6 )-Ib is most closely related to aac(6 )-IIa, aac(6 )-IIb, aac(6 )-IIc, and aac(6 )-IId.

Ethics Statement
Biomedical Ethics Committee of the 'Institut Pasteur de Tunis' approved this work, and the sample technique was carried out in accordance with the widely accepted ISO 10272-1:2006 Appendix 2 standards for the identification of Campylobacter spp.

Extraction of Bacterial DNA
The genomic DNA of collected isolates was extracted using the boiling method [32].Briefly, Campylobacter isolates were grown in 2 mL Bolton broth (Oxoid, United Kingdom) and plated on Karmali agar (SIGMA-Aldrich, Germany).Campylobacter colonies were then harvested and suspended in 100 µL TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0).Cell suspensions were heated at 100 • C for 10 min and then cooled at −20 • C. Thereafter, cell suspensions were centrifuged at 8000 rpm for 5 min [32].The supernatant containing DNA was collected, transferred into a new tube, and then stored at −20 • C until use.

Detection of Mutation in the QRDR of gyrA via Mismatch Amplification Mutation Assay (MAMA-PCR)
The quinolone region determining resistance (QRDR) of the GyrA subunit of the ADN gyrase enzyme is where the fluoroquinolone resistance is often codified by a point mutation (Thr-86-Ile) [37].A single-point mutation Thr-86-Ile in the quinolone resistance-determining region (QRDR) of the gyrA gene was defined as the main mechanism of high-level resistance to fluoroquinolones.Campylobacter isolates were subjected to analysis via MAMA-PCR, as previously described for C. jejuni and C. coli isolates [37,38]  Positive PCR products in selected five isolates were directly sequenced to identify the aac(6)-Ib gene.Nucleotide sequencing was carried out with an Applied Biosystems™ 3500 sequencer calibrated with BigDye ® Terminator V3.1.DNA sequences were assembled and edited using BioEdit Sequence Alignment Editor software version 7.0.5.3.The consensus sequences obtained were compared to sequences in the Basic Local Alignment Search tool (BLAST).The sequences generated in this study were deposited in GenBank under the following accession numbers: OR124788, OR125068, OR125070, OR125069, and OR192836.

Molecular Phylogenetic Analysis via the Maximum Likelihood Method
The evolutionary history was inferred using the maximum likelihood method based on the Kimura two-parameter model [39].The tree with the highest log likelihood (−1721.35) is shown.The percentage of trees in which the associated taxa clustered together is shown next to the branches.Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach, and then selecting the topology with a superior log likelihood value.A discrete Gamma distribution was used to model evolutionary rate differences among sites (five categories (+G, parameter = 200.0000)).The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 8.19% sites).The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.The analysis involved 34 nucleotide sequences.All positions containing gaps and missing data were eliminated.There were a total of 421 positions in the final dataset.Evolutionary analyses were conducted in MEGA7 [40].

Data Analysis
All collected data were analyzed using R software (www.infostat.com.ar/accessed 24 July 2023).The antimicrobial resistance analyses were performed by means of a Chisquare statistic (p < 0.05) [41].This test is a nonparametric tool designed to compare frequency counts between two groups of different sample sizes; the selection criterion for significantly prevalent variance was a stringent p-value of 0.001 or less.The phylogenetic tree was generated using iTOL version 6 (https://itol.embl.de/accessed 5 September 2023).The strains of our study are reported using their accession number code displayed in bold.

Conclusions
The results of this study showed a significant prevalence of aac(6 )-Ib and aac(6 )-Ib-cr, as well as a high rate of co-carriage of these two genes among Campylobacter isolates in Tunisia.It seems that quinolone/fluoroquinolone resistance is established, mainly in C. coli, by chromosomal mutations the gyrA gene, and evolves to acquire aac(6 )-Ib-cr against a genetic background already rich in wild-type aac(6 )-Ib gene.

Figure 2 .
Figure 2. Molecular phylogenetic analysis by maximum likelihood method.The evolutionary history was inferred by using the maximum likelihood method based on the Kimura two-parameter model.The tree with the highest log likelihood (−1721.35) is shown.The percentage of trees in which the associated taxa clustered together is shown next to the branches.Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach, and then selecting the topology with superior log likelihood value.A discrete Gamma distribution was used to model evolutionary rate differences among sites (five5 categories (+G, parameter = 200.0000)).The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 8.19% sites).The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.The analysis involved 34 nucleotide sequences.All positions containing gaps and missing data were eliminated.There were a total of 421 positions in the final dataset.Evolutionary analyses were conducted in MEGA7.ITOL version 6 was used to create the phylogenetic tree.The strains in our investigation are described using their bolded accession number code. .

Table 1 .
Percentage of resistant Campylobacter isolates.