Prevalence, molecular characterization of integrons and its associated gene cassettes in Klebsiella pneumoniae and K. oxytoca recovered from diverse environmental matrices

The high prevalence of infections arising from Klebsiella species is related to their ability to acquire and disseminate exogenous genes associated with mobile genetic elements such as integrons. We assessed the prevalence, diversity, and associated gene cassettes (GCs) of integrons in Klebsiella species. The isolates recovered from wastewater and hospital effluents, rivers, and animal droppings were identified using the conventional Polymerase Chain Reaction (PCR) with primers targeting the gryA, pehX, and 16S–23S genes. The antimicrobial resistance profile and the Extended-Spectrum and Metallo β-lactamases production were carried out using standard microbiological techniques. PCR, DNA sequencing analyses, and Restriction Fragment Length Polymorphism were used to characterize the integrons and their associated GCs. Furthermore, the genotypic relationships between the different isolated K. pneumoniae were determined using Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR. About 98% (51/52) of the confirmed isolates harboured an integrase gene, with 80% intI1, while the remaining 20% concurrently harboured intI1 and intI2, with no intI3 observed. About 78% (40/51) of the bacterial strains were positive for a promoter, the P2R2, investigated, while 80% (41/51) harboured at least one of the qacEΔ1 and sul1. Three different GCs arrangements identified were aac(6′)-Ib, aadA1-dfrA1, and dfrA1-sat2. At a similarity index of 60%, the ERIC-PCR fingerprints generated were categorized into nine clusters. Our study is the first to reveal the features of integrons in Klebsiella spp. recovered from environmental sources in the Eastern Cape Province, South Africa. We conclude that the organisms' sources are repositories of integrons harbouring various gene cassettes, which can be readily mobilized to other microorganisms in similar or varied niches.

PCR-based confirmation of Klebsiella spp..The molecular confirmation of the Klebsiella genus was by the conventional Polymerase Chain Reaction (PCR) assay using primer sets that target the gryA gene.All genus-confirmed Klebsiella isolates were speciated into K. pneumoniae and K. oxytoca using primers targeting the 16S-23S ITS and pehX genes, respectively.K. oxytoca NCTC 11686 was included as a positive control (Microbiologics, Medimark, France), while E. coli ATCC 8739 was used as the negative control following conditions described in Table 1.
The inoculum of Klebsiella spp. was suspended in a sterile saline solution with the turbidity adjusted to the 0.5 McFarland standard and evenly spread on Mueller-Hinton agar plates (Oxoid, UK).A disc dispenser (Mast Diagnostics, South Africa) was used to place the antibiotics.The plates were incubated at 37 °C for 18 h.After that, the inhibition zones' width was measured to the nearest millimetre and compared to the CLSI-established breakpoints to categorize the isolates as resistant, intermediate, or susceptible.Isolates resistant to more than two antimicrobial classes were considered MDR 45 .
The ethylenediaminetetraacetic acid (EDTA) test was used to determine the production of metallo β-lactamase (MBL) according to CLSI standards.A 0.5 McFarland adjusted test isolate was exposed to two 10 µg imipenem discs.Then, 10 µl of 0.5 M EDTA was added to only one disc to obtain a concentration of 750 µg, and an increase in inhibitory zone width of 5 mm in the disc potentiated with EDTA after 18 h of incubation at 37 °C was recorded as positive for MBL generation 45 .

Molecular detection and characterization of integrons.
The confirmed bacterial strains were screened with conventional PCR assays as described above.The presence of intI1, intI2, and intI3 located on the 5′-CS was assayed for the classification of the integrons.In addition, P2R2 on the 5′-CS of a typical integron was also screened to detect the presence of a promoter.Furthermore, the intI1-positive strains were assessed for genes (qacEΔ1 and sulI) on the ORFs at the 3′-CS of a typical class 1 integron.The positive control for a typical class 1 integron used was Acinetobacter baumannii ATCC 19606.All primers and thermal cycling conditions are listed in Table 1.

Mapping of integrons.
In separate PCR assays, isolates positive for the intI1 and intI2 were assessed to detect their internal variable regions (IVRs).Specific primer set 5′-CS and 3′-CS, which joins the attI1 site of the 5′-CS and the 3′-CS of intI1, was used for intI1 positive isolates while the primer set hep74 and hep 51, which binds to the attI2 and the orfX sites located downstream the GC regions, was used for intI2 positive isolates, as indicated in Table 1.The PCR assays were carried out in triplicates to ensure reproducibility.www.nature.com/scientificreports/Restriction analysis and DNA sequencing of amplicons.Amplicons of variable regions that appeared similar in size were exposed to the AluI restriction enzyme (Biolabs, England) for the restriction fragment length polymorphism (RFLP) to assess if the products were in the same sequence.For the identification of similar GC arrays in the integrons, AluI was chosen due to its recognition sequence being only four bases, thereby increasing the likelihood of its activity over the other enzymes that target the six-base sequence.Briefly, 30 µl of the amplicon was exposed to 1.0 µl of 10U/ml AluI.The reaction mixture was incubated for 4 h at 37 °C.The products were then run on a 2% agarose gel and visualized.These were characterized according to their distinct restriction profiles, and two randomly selected representative amplicons from each RFLP class were selected for sequencing.This step was taken to help reduce the risk of needlessly sequencing multiple identical variable regions.
Variable sequence analysis, cassette identification.The content and arrangement of the inserted GCs within the amplified IVRs were analyzed through sequencing.The amplicons were sequenced in both directions on an ABI 3500XL sequencer using the Nimagen BrilliantDye™ Terminator Cycle sequencing kit V3.1 (Inqaba Biotechnical Industries, South Africa).The sequences were then modified with Chromas 2.7 and pairwise aligned using BioEdit sequence alignment editor software.BLAST nucleotide search analysis (https:// blast.ncbi.nlm.nih.gov/ Blast) was performed on the generated consensus sequences to identify the contents of the inserted gene cassette.The position of each gene in each cassette was determined using ABRicate 0.8.4 (https:// github.com/ tseem ann/ abric ate) (ResFinder, ARG-ANNOT, CARD, and NCBI databases).
The amplicons were resolved in 3% agarose gel in a 0.5X TBE buffer and allowed to run at 100 V for 240 min.

Statistical analysis.
The descriptive statistical software in Microsoft Excel 2016 and the Statistical Package for the Social Sciences (SPSS) version 25 were used to examine the data (SPSS Inc., Chicago, IL).The data was validated, and relationships were calculated with 2 × 2 cross-tabulation tables utilizing exploratory data analysis.
Pearson's Chi-square test was used to examine the statistical significance of susceptibility and the number of integron-positive isolates.A P value of less than 0.05 was considered significant.Using Gelj v.2.0 software, computer-assisted pattern analysis was used to examine the DNA fingerprints generated from the ERIC-PCR.Pearson's correlation coefficient was used to compute the percentage similarity of digitized bands.The relatedness of the isolates was estimated using the unweighted pair group method with arithmetic mean (UPGMA) and complete linkage methods, which were shown as dendrograms.

Results
PCR confirmation of bacterial strains.From the 52 isolates that were positive for the gryA gene to confirm Klebsiella spp., 63% (33/52) were positive for 16S-23S ITS to confirm K. pneumoniae.About 37% (19/52) of the remaining isolates were positive for the polygalacturonase pehX gene and identified as K. oxytoca.

Characterization of 5′ conserved segment (integrase + promoters). Among the 52 confirmed
Klebsiella spp., 98% (51/52) were integron-positive as they harboured the integron integrase (intI1) gene.All the K. pneumoniae harboured the intI1 gene.The intI1 gene was absent in only one K. oxytoca isolate, while 20% (10/51) of these intI1 positive isolates concurrently harboured the intI2 gene and were thus classified as class 1 + 2 integrons.Those carrying only the intI1 gene classified as class 1 integrons were 80% (41/51).Notably, none of the isolates harboured the intI2 gene only, as when present, they concurrently were found with intI1.No intI3 gene for class 3 integrons was detected (Fig. 1).As shown in Table 2, detecting the integrase gene in each isolate was associated with resistance to three antibiotics, ciprofloxacin, amoxicillin-clavulanic acid, and imipenem at significant levels (P < 0.05).

Characterization of internal variable regions (IVRs). The internal variable regions that harbour GCs
and are between the CS of the integron harbouring isolates were 53% (27/51).The mapping of intI1 and intI2 positive isolates revealed that 59% (22/37) and 50% (5/10) of the integrons were positive for IVRs, respectively.The K. oxytoca isolates with class 1 + 2 integrons did not harbour any IVRs.The amplified IVRs ranged from 160 to 1400 bp, with the mode being the least base pair.The analysis of the IVRs yielded eight amplicons of distinctly varied sizes as follows: ≈ 160 bp (n = 18), ≈ 190 bp (n = 2), ≈ 280 (n = 2), ≈ 350 (n = 1), ≈ 550 (n = 1), ≈ 700 bp (n = 1), ≈ 1200 (n = 1), and ≈ 1400 (n = 1).The analysis of the sequences yielded three distinct cassette arrays.These detected cassette arrays were aac(6′)-Ib, aadA1-dfrA1, and dfrA1-sat2 (Table 3).They harboured genes that encode resistance to aminoglycosides, trimethoprim, and streptothricin.The most detected cassette was aac(6′)-Ib representing 82% (18/22) of all identified cassettes.The gene dfrA1 was detected in 9% of the cassettes, while sat2 and aadA1 occurred only once.The sequences of the remaining IVR amplicons yielded empty or undetermined cassette arrays indicating they are likely to be novel integrons or integrons without any GCs, as they did not yield meaningful alignment when compared with the sequences in the GenBank.The schematic representation of some of the integrons and their associated gene cassette arrays are shown in Fig. 2.

Genetic relatedness of K. pneumoniae from various sources.
The genotypic relatedness of all the K. pneumoniae investigated is indicated in the dendrogram generated from the ERIC-PCR fingerprinting of isolates.The fingerprints generated nine clusters (Fig. 3A-I) at a similarity cut-off value of 60%.Cluster I was the highest ERIC-genotype cluster.It comprises seven isolates with representatives from various water sources, with similarities ranging from 67 to 78%.Cluster C was grouped with five isolates from HWW and WWTP, while the least ERIC-genotype cluster was observed at F and G (one isolate from HWW and river, respectively).The eight FD isolates were spread into three clusters, three clustered at A while four clustered at E with 68% and 65% similarity indices.Cluster D was formed with an isolate each from the river and FD with 63% similarity.Cluster B contained two isolates from WWTP, while cluster H contained an isolate from WWTP, HWW, and river.It www.nature.com/scientificreports/should be noted that 5 of the K. pneumoniae isolates did not generate any visible bands on the ERIC-PCR gel and were excluded from the analysis.

Discussion
On speciation of the Klebsiella investigated in this study, K. pneumoniae (63%) was more frequently detected than K. oxytoca (37%).It agrees with the reports that K. pneumoniae is the most prevalent species of the genus as it is the causative agent of most nosocomial Klebsiella infections, thereby referred to as the most medically significant species of the genus 3,47 .K. pneumoniae is becoming more recognized as an invasive and aggressive pathogen that carries some ARGs 48 .It has been reported to cause fatalities in various provinces within South Africa 49,50 .Antibiotic resistance was shown to be prevalent among the isolates in this investigation.They were all categorized as MDR because they were resistant to more than two distinct classes of antibiotics (Fig. 1).Many antibiotics are not entirely metabolized after consumption and end up in wastewater systems 34 .Antibiotics in wastewater have been demonstrated to impose selection pressure on antimicrobial-resistant bacteria, allowing them to spread to new environments 51 .This widespread usage of antimicrobials has resulted in an increased rate of antimicrobial resistance detection in bacteria.Almost all the isolates in this study were resistant to the β-lactams, including the third-generation cephalosporins.The least resistance was observed to imipenem, which is not usually a front-line prescription antibiotic.This low detection rate in resistance to carbapenems is somewhat expected and similar to reports of other studies where the resistance frequencies ranged from 7 to 15% 52,53 .MDR K. pneumoniae has emerged as a critical problem in treating nosocomial infections worldwide 2 ; therefore, its environmental detection is quite concerning.The production of β-lactamase by Klebsiella spp. is one of their resistance mechanisms to β-lactam antibiotics 54 .Our study revealed a prevalence phenotype of 50% and 12% of ESBL and MBL, respectively.These β-lactamases are also associated with co-resistance to other antibiotics www.nature.com/scientificreports/by bacterial species harbouring them, further limiting possible treatment options.These β-lactamases could be carried on mobile genetic elements such as integrons, which aid in the horizontal spread of ARGs among diverse bacterial species 48,54,55 .In a study in China, clinically relevant strains of ESBL-producing K. pneumoniae were also found in diverse environmental sources 7 .They also reported that all the isolates were MDR, with a few being clinically relevant strains known to cause hospital outbreaks.These studies, including ours, aim to draw our attention to the need to ascertain the involvement of the environment in the spread of these pathogens and the concomitant risk to public health.In this study, integrons were detected in all but one of the isolates investigated, with class 1 integrons being most predominant.The studies of 2 and 18 reported that all their MDR K. pneumoniae harboured the intI gene, although these isolates were from clinical samples.Other research from environmental origins has also reported the detection of the intI1 gene as the most prevalent in similar studies 13,22,56 .Furthermore, the predominance of intI1 in this study is similar to other studies reporting that class 1 integrons are more prevalent than class 2 integrons in Gram-negative bacteria 31,[56][57][58][59] .There is a strong association between the occurrence of integrons and the prevalence of MDR in Gram-negative bacteria due to their high capacity for transferring antimicrobial resistance genes 31,56,60 .Although our results indicate a significant association between intI1 and three out of thirteen antibiotics investigated (Table 3), in research by Li and colleagues 61 , integron harbouring isolates demonstrated resistance to a substantially greater number of antibiotics than negative isolates.Other investigations have found a high prevalence of integron-positive MDR Klebsiella spp. 2,62.Integrons give a selective advantage to bacteria in settings where antibiotic use causes selective pressures, which may explain the high occurrence of integrons among MDR strains.
Typical class 1 integrons usually harbour quaternary ammonium compounds and sulphonamide resistance genes 20,63 .In this study, 59% (30/51) of the integrase-positive isolates harboured both sul1 and qacEΔ1, further confirming the presence of the 3′ CS of a typical class 1 integron which was also reported in similar studies 56,57 .It was observed that 27% (8/30) of these isolates concurrently have the intI2 gene (Fig. 1).Invariably, this shows that it is possible that some bacteria with a typical class 1 integron can still harbour the class 2 integrase gene, a scenario that has been previously reported 56 .Furthermore, we report a prevalence of 78% of a promoter type, the P2R2, located on the integron.It further confirms that an integron can act as an expression vector as this promoter can be involved in the expression of the genes in the cassette arrays acquired from the environment once incorporated into the integrons.The inability to detect the P2R2 on the remaining isolates via PCR could have been due to a mutation in the promoter gene located within the integrase, or those integrons could have a promoter type different from the assayed gene.
Three unique GC arrays in the integrons are shown in Table 2.These results prove that environmental sources also serve as a potential repertoire of mobile genetic elements harbouring different antibiotic resistance GCs.The GCs contained genes encoding resistance to trimethoprim (dfrA1), streptothricin (sat2), and aminoglycosides (aadA1 and aac( 6)-Ib).The most prevalent GC was the aac(6)-Ib, which occurred singly.Our results indicate that 53% (27/51) of the isolates harbouring the intI1 contained the internal variable regions (Fig. 1).The aac(6)-Ib encodes an enzyme 6′-N-acetyltransferase and belongs to the family of aminoglycoside acetyltransferases, while the aadA1 encodes the aminoglycoside adenyl transferase.They confer resistance to aminoglycosides such as amikacin, gentamicin, tobramycin, neomycin, and streptomycin.The Sat2 encodes streptothricin-Nacetyltransferase, which confers resistance to streptothricin.The detection of the dfrA encodes dihydrofolate reductase, which confers resistance to trimethoprim, has been associated with selection pressure and high use of trimethoprim, especially in clinical sources 2 .However, this study infers that such a selective burden may equally exist in contaminated aquatic environments.
According to the sequencing results, 13.7% of these isolates did not contain any GCs (Table 3), and these could be a result of defects or mutations at the 3′ CS or the GCs belonging to an unusual or complex class 1 integrons 31,64 .The detection of integrons with empty GCs has been reported, and it shows the readiness of such isolates to capture GCs readily and subsequently express the ARGs harboured on them.The ERIC-PCR dendrogram image, as seen in Fig. 3, shows the clonal relatedness of the integron-positive K. pneumoniae from the various sources clustered in similar groups, indicating high genetic relatedness among the integron-bearing MDR isolates.This further reiterates the ability of bacterial species from various niches to exchange genetic elements, which can confer specific adaptability properties.

Conclusions
MDR bacterial strains have been widely disseminated due to the distribution of antibiotic-resistant strains, particularly the ESBL and carbapenemase producers.The occurrence of integrons in these MDR bacterial strains has further accelerated the spread of ARGs into diverse environmental sources.The data obtained from this study show that integrons with their associated gene cassettes are widely distributed in Klebsiella spp.from environmental matrices that may further constitute a problem when treating bacterial infections.The baseline properties of integrons in Klebsiella species isolated from several environmental matrices in the Eastern Cape Province, South Africa, have never been previously reported.

Figure 2 .
Figure 2. Schematic representation of some integron-positive K. pneumoniae with their associated gene cassette arrays.These isolates were positive for intI1 and intI1 + 2 for class 1 and class 1 + 2 integrons, respectively.The P2R2 for the promoter is situated on the 5′ conserved segment.Also detected were sul1 and qacEΔ1 genes found on the 3′ conserved segment conferring resistance to sulphonamides and quaternary ammonium compounds, respectively.The cassette arrays include the aadA1 and aac(6′)-Ib gene, which encodes resistance to aminoglycosides.The dfrA1 and sat2 confer resistance to trimethoprim and streptothricin, respectively.The start and stop nucleotide sequences were also depicted, while the arrows indicate the transcription direction.

Figure 3 .
Figure 3. UPGMA dendrogram image obtained from clustering analysis indicating the genetic relatedness of K. pneumoniae (n = 28) recovered from various environmental sources using the ERIC-PCR technique.HWW Hospital wastewater, WWTP wastewater treatment plant, FD animal droppings.

Table 1 .
The list of primers and thermocycling conditions used for PCR amplification.

Table 2 .
The association between resistance to antibiotics and the presence of integrons in Klebsiella spp.Significant values are represented in bold.a No statistics are computed because all the isolates displayed a 100% resistance to Nalidixic acid.

Table 3 .
The content and arrangement of genes within the integrons' internal variable regions (n = 27).
a Except for three isolates that were positive for qacEΔ1 only, one isolate was positive for sul1 only, while three of the isolates lacked both qacEΔ1 + sul1.bTheamplicon yielded an undetermined/empty cassette array.c One isolate lacked both qacEΔ1 + sul1.