https://orcid.org/0000-0001-9602-9630
Abstract
Actinobacillus pleuropneumoniae infection causes a high mortality rate in porcine animals. Antimicrobial resistance poses global threats to public health. The current study aimed to determine the antimicrobial susceptibilities and probe the resistome of A. pleuropneumoniae in Taiwan. Herein, 133 isolates were retrospectively collected; upon initial screening, 38 samples were subjected to next-generation sequencing (NGS). Over the period 2017–2022, the lowest frequencies of resistant isolates were found for ceftiofur, cephalexin, cephalothin, and enrofloxacin, while the highest frequencies of resistant isolates were found for oxytetracycline, streptomycin, doxycycline, ampicillin, amoxicillin, kanamycin, and florfenicol. Furthermore, most isolates (71.4%) showed multiple drug resistance. NGS-based resistome analysis revealed aminoglycoside- and tetracycline-related genes at the highest prevalence, followed by genes related to beta-lactam, sulfamethoxazole, florphenicol, and macrolide. A plasmid replicon (repUS47) and insertion sequences (IS10R and ISVAp11) were identified in resistant isolates. Notably, the multiple resistance roles of the insertion sequence IS10R were widely proposed in human medicine; however, this is the first time IS10R has been reported in veterinary medicine. Concordance analysis revealed a high consistency of phenotypic and genotypic susceptibility to florphenicol, tilmicosin, doxycycline, and oxytetracycline. The current study reports the antimicrobial characterization of A. pleuropneumoniae for the first time in Taiwan using NGS.
Introduction
Actinobacillus pleuropneumoniae is a gram-negative, rod-shaped, facultatively anaerobic bacterium. It usually causes porcine pleuropneumonia with a high mortality rate, thereby leading to tremendous economic losses in the swine industry worldwide (Sassu et al. Citation2018). To date, 19 serovars of A. pleuropneumoniae have been recognized (serotypes 1 to 19), which differ in pathogenicity and might exhibit different biological behaviors (Stringer et al. Citation2021). Depending on the requirement for nicotinamide adenine dinucleotide (NAD) for growth, A. pleuropneumoniae can be classified as two biovars, namely, biovar I (typical) and biovar II (atypical). Biovar I is NAD-dependent, while biovar II is NAD-independent. Normally, biovar I contains serovars 1–12, 15, and 16, and serovars 13 and 14 belong to biovar II. Nevertheless, some unclassified isolates were also proposed (Ito et al. Citation2016). A. pleuropneumoniae shows considerable geographic variation and differences in dominant biovars/serovars among countries. For instance, serovars 1 and 5 are common in North and South America (Dubreuil et al. Citation2000), and serovars 5 and 7 are the most frequent serotypes in Canada (Lacouture and Gottschalk Citation2020). Serovar 2 is predominant in Europe (Dubreuil et al. Citation2000; Schuwerk et al. Citation2021) and the second most prevalent serotype in the Czech Republic (Kucerova et al. Citation2018). Serotyping of A. pleuropneumoniae is determined by an antibody-based test, based on the presence of different capsular polysaccharides. With increased accuracy and reproducibility compared to these traditional methods, studies have increasingly switched to molecular serotyping (Gottschalk and Lacouture Citation2015). Vaccination and use of antimicrobials are common in A. pleuropneumoniae prevention and treatment (Sassu et al. Citation2018). Although several clinical trials have used vaccines to control A. pleuropneumoniae, the limited protection against this disease restricts further investigations (Ramjeet et al. Citation2008; Stringer et al. Citation2021). Furthermore, a well-designed vaccine that provides protection against all serotypes is still under development (Ramjeet et al. Citation2008). Thus, the most common and effective medical intervention is the administration of antimicrobial agents. However, numerous studies have demonstrated acquired resistance to tetracycline in A. pleuropneumoniae (Kucerova et al. Citation2011; Bossé et al. Citation2017). Vanni et al. (Citation2012) reported finding increasing levels of resistance to penicillin, amoxicillin, ampicillin, and macrolides. These results highlighted the unmet needs for disease control and a complete understanding of antimicrobial resistance (AMR) in A. pleuropneumoniae.
The prevalent use of antimicrobial agents to combat A. pleuropneumoniae leads to the emergence of resistant A. pleuropneumoniae isolates. Typically, antimicrobial susceptibility testing (AST) is conducted by disk diffusion or minimum inhibitory concentration (MIC) assays (Bossé et al. Citation2017). Owing to its convenience, AST via disk dilution is one of the most common methods and is regularly employed in the field. However, this strategy might provide insufficient information on disease control since the long-term outbreaks of A. pleuropneumoniae are retained. Furthermore, molecular behaviors, AMR gene patterns, and evolutionary degree cannot be reliably inferred from this test. Whole-genome sequencing (WGS) offers robust tools to overcome these difficulties, which have been proposed to evaluate the microbiome and resistome of the isolates. Surveillance for AMR genes (ARGs) using WGS can be useful, as WGS is a sensitive method for detecting known ARGs in A. pleuropneumoniae (Cohen et al. Citation2021) and multiple ARGs that can contribute resistance to a certain antibiotic (Bossé et al. Citation2017). Therefore, applying next-generation sequencing (NGS) to probe the data from WGS would help provide a comprehensive understanding of the resistance patterns of bacterial isolates (Gajic et al. Citation2022). Comparing genome sequence data offers extensive molecular and epidemiologic information. Previous studies reported high concordance between the resistance profiles obtained using NGS and those obtained using phenotypic ASTs, which demonstrated that results from genome sequences are highly correlated with phenotypic resistance (Faksri et al. Citation2019; Hendriksen et al. Citation2019). Furthermore, the mechanisms and pathways involved in serotypes remain unclear, and the advent of NGS offered powerful tools to reveal potential mechanisms (Besser et al. Citation2018).
In veterinary medicine, NGS has gradually developed for epidemiological studies, the surveillance of antibiotic resistance, and the detection of virulence genes (Donà et al. Citation2022; Kardos et al. Citation2022). However, few studies have reported resistant A. pleuropneumoniae isolates detected through comprehensive phenotypic and genotypic analysis. Although ASTs have been regularly employed in the clinic (Bayot and Bragg Citation2023), ineradicable AMR and multiple drug resistance (MDR) remain tremendous challenges in Taiwan’s porcine industry. Therefore, the purpose of the current study was to report the prevalence and AMR patterns of A. pleuropneumoniae infection in Taiwan from 2017 to 2022. Furthermore, the present study documented the widespread presence of phenotypic and genotypic resistomes in pigs infected with A. pleuropneumoniae.
Materials and methods
A. pleuropneumoniae isolation
The investigated A. pleuropneumoniae isolates were collected from 2017 to 2022 at the Animal Disease Diagnostic Center, National Pingtung University of Science and Technology, Taiwan. The 133 samples were collected from 84 different husbandry farms. The animals had not been treated with antimicrobial agents prior to sample collection. A. pleuropneumoniae isolates were isolated overnight on chocolate agar plates supplemented with PolyViteX (bioMérieux, Marcy l’Etoile, France) at 37 °C with 5% CO2. All bacterial strains were immediately subcultured on the same culture medium used for the primary isolation and submitted to ASTs. An A. pleuropneumoniae-specific PCR was employed for species identification (Chiers et al. Citation2001).
Susceptibility of A. pleuropneumoniae isolates
Antibiotic susceptibility was determined by the disk diffusion method. The following 12 antimicrobials that were commonly recommended and administered to treat respiratory diseases were selected, namely, trimethoprim/sulfamethoxazole (TMP/SMX), amoxicillin (AMO), ampicillin (AMP), enrofloxacin (ENR), cephalexin (CFX), cephalothin (CTN), ceftiofur (CEF), doxycycline (DOX), oxytetracycline (OTC), florfenicol (FFC), gentamicin (GEN), tilmicosin (TIL), streptomycin (STR), and kanamycin (KAN). The results of ASTs were interpreted by the breakpoints defined by the Clinical and Laboratory Standards Institute 2023 standards (CLSI VET01SED6: 2023 Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals, 6th Edition, shows in ) or previous studies (Yang et al. Citation2011). The antibiotic groups were classified according to a previous study with simple modification: beta-lactam, tetracyclines, quinolones and fluoroquinolones, cephalosporins, aminoglycosides, phenicols, macrolides, and trimethoprim-sulfamethoxazole (Korytny et al. Citation2016).
Extraction of genomic DNA and preparation of DNA sequencing libraries
Total genomic DNA was isolated from each sample using a DNA Extraction Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. A Qubit fluorometer was used to measure the concentrations of genomic DNA following the standard protocol (Invitrogen, Carlsbad, CA, USA).
For library preparation, all reagents used were provided in the Illumina commercial kits. The Illumina DNA Preparation Kit (Illumina, San Diego, CA, USA) and IDT for Illumina DNA/RNA/UD indexes Set A (Illumina) were employed for sequencing library preparation following by the manufacturer’s instructions. The quality of the indexed libraries was determined in ng/μL units on a Qubit fluorometer (Invitrogen). Quantitation and visualization were performed using capillary electrophoresis (Bioptic, New Taipei City, Taiwan). The average base pair for each peak location was determined for the samples. Using the formula recommended by Illumina, concentrations were converted to nM values. Each DNA library was normalized and diluted to a final loading concentration of 100 pM (Sahin et al. Citation2021). The pooled DNA libraries were loaded onto the iSeq 100 flow cell (Illumina) for sequencing in a total volume of 20 μL. The cartridge (with the preinserted flow cell) was then inserted into the iSeq 100 sequencer (Illumina). Whole-genome sequencing was performed on the sequencer with an approximately 17-h run time using paired-end sequencing of 2 × 150 bp reads.
iSeq 100 QC and NGS bioinformatic analysis
Sample quality control (QC) metrics of the raw reads were first calculated using DRAGEN FastQC plus MultiQC. The QC reports were generated for all samples. After passing QC, the raw reads were trimmed by Trimmomatic v.0.36 software to remove reads with technical bias, low-quality reads, and adapters (Bolger et al. Citation2014). All raw reads were de novo assembled using SPAdes Genome Assembler v3.9.0 in auto K-mer mode (Bankevich et al. Citation2012). The assembled files were then subjected to gap closing using Rescaf software. The gap-filled assemblies were annotated via various bioinformatic tools.
KmerFinder 3.2 was employed based on K-mer statistics for identifying bacterial species in WGS data (Clausen et al. Citation2018). Assemblies were annotated using ResFinder v.4.3.3 (Florensa et al. Citation2022) for resistome analysis. The gene presences were determined with the setting >80% hit length and >98% sequence identity. KmerResistance 2.2 examines the co-occurrence of K-mers between WGS data and a database of resistance genes. This tool was applied to identify acquired antibiotic resistance genes using K-mers (Clausen et al. Citation2018). MobileElementFinder v.1.0.3 identified mobile genetic elements and their relation to antimicrobial resistance genes and virulence factors (Johansson et al. Citation2021). Phylogenetic trees were inferred with NDtree 1.2 (https://cge.food.dtu.dk/services/NDtree/, accessed on 3 September 2023) (Leekitcharoenphon et al. Citation2014) and further visualized using the Interactive Tree of Life (iTOL v5) web server (https://itol.embl.de/, accessed on 3 September 2023) (Letunic and Bork Citation2021).
Raw next-generation sequencing data generated in this study has been deposited to the publicly accessible NCBI Sequence Read Archive (SRA) under the accession numbers: PRJNA1045321 (SAMN38440904 - SAMN38440923) and PRJNA1045325 (SAMN38441020 - SAMN38441037).
Statistical analysis
Statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA). The Fisher’s exact test was employed to determine whether there is a significant association between two categorical variables. The statistical difference was defined as a p value below 0.05. A kappa value between 0 and 0.2 is considered slight agreement, 0.2 to 0.4 fair, 0.4 to 0.6 moderate, 0.6 to 0.8 substantial, and > 0.8 almost perfect agreement (Audigé Citation2005).
Results
Prevalence of antimicrobial resistance from 2017 to 2022
The susceptibility of 133 A. pleuropneumoniae clinical isolates was assessed against 12 commonly used antibacterial agents. The resistance rates and resistance to individual antimicrobials of A. pleuropneumoniae isolated from 2017 to 2022 are summarized in and , respectively. Isolates showed a higher resistance rate for tetracyclines (columns in blue, oxytetracycline and doxycycline), beta-lactams (columns in red, amoxicillin and ampicillin), and aminoglycosides (columns in purple, streptomycin, kanamycin, and gentamicin) than for the other antimicrobials. Notably, from 2021 to 2022, a significant decrease was found for amoxicillin (from 68.6% to 33.3%, p = .0071) and ampicillin (from 77.1% to 33.3%, p = .0005), whereas the oxytetracycline resistance rate was significantly increased (from 77.1% to 100.0%, p = .0051). When comparing the resistance rate between years 2017 and 2022, the decreased trends were observed for amoxicillin (from 61.5% to 33.3%, p = .1042), ampicillin (from 61.5% to 33.3%, p = .1042), enrofloxacin (from 15.4% to 6.1%, p = .5654), gentamicin (46.2% to 15.2%, p = .0509), and trimethoprim-sulfamethoxazole (from 38.5% to 9.1%, p = .0306). Isolates showed an increased resistance rate for streptomycin (from 46.2% to 93.9%, p = .0009) and kanamycin (from 38.5% to 63.6%, p = .1873), whereas relatively few changes were observed for doxycycline (from 76.9% to 87.9%, p = .1042), oxytetracycline (from 92.3% to 100.0%, p = .1042), cephalothin (from 0.0% to 6.1%, p > .9999), cephalexin (from 7.7% to 0.0%, p = .2826), ceftiofur (from 7.7% to 0.0%, p = .2826), florfenicol (from 53.8% to 42.4%, p = .5274), and tilmicosin (from 15.4% to 19.0%, p > .9999). Furthermore, antimicrobial resistance rates of enrofloxacin (6.0%) and cephalosporins (from 0.8% to 4.5%) were relatively low compared with other agents (, resistance rate in total). These findings suggest that cephalosporins (cephalothin, cephalexin, and ceftiofur) and enrofloxacin were more effective than other antibiotic choices for the control of A. pleuropneumoniae.
Figure 1. Occurrences of antimicrobial resistance in Actinobacillus pleuropneumoniae isolates. (A) Resistance rates of A. pleuropneumoniae against commonly used antimicrobials during 2017–2022. (B–G) Distribution of antimicrobial resistance in A. pleuropneumoniae isolates in the separate years (2017–2022). Tetracyclines (OTC and DOX) are shown as blue bars. Red bars represent penicillins (beta-lactam; AMO and AMP). Green bars indicate cephalosporins (CTN, CFX, and CEF). Aminoglycosides, including streptomycin (STR), kanamycin (KAN), and gentamycin (GEN), are shown in purple. Other columns in one color represent certain antimicrobials (FFC, GEN, TMP/SMX, and TIL). Numbers indicate the percentages of cases. OTC: oxytetracycline; DOX: doxycycline; AMP: ampicillin; AMO: amoxicillin; FFC: florfenicol; GEN: gentamycin; TMP/SMX: trimethoprim/sulfamethoxazole; TIL: tilmicosin; ENR: enrofloxacin; CTN: cephalothin; CFX: cephalexin; CEF: ceftiofur.
Table 1. Resistance breakpoints detected using the disk diffusion method for Actinobacillus pleuropneumoniae based on the previous studies and/or the clinical and laboratory Standards institute 2023 standards (CLSI VET01 ED6: 2023 Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals, 6th Edition).
Table 2. Antimicrobial resistance patterns of Actinobacillus pleuropneumoniae isolates against commonly used antimicrobials during 2017–2022.
Multiple-drug-resistance patterns in Taiwan
MDR poses a global threat to public health. Herein, we evaluated the antimicrobial resistance patterns in A. pleuropneumoniae isolates. In total, 73 AMR profiles were identified among 133 A. pleuropneumoniae isolates (Table S1). Of the 133 isolates, 95 (71.4%) samples were identified where the isolates showed resistance to three or more antimicrobial categories (, MDR profiles). Isolates with resistance to four (39.1%) types of drugs were the most common, followed by those with resistance to two (24.8%) and three (21.8%) drugs. As shown in , the most common antimicrobial resistance pattern was ‘AMO-AMP-DOX-OTC-FFC-STR-KAN’, and there were eight isolates with this phenotype. These results indicated the severe MDR of A. pleuropneumoniae in Taiwan.
Figure 2. Percentage of Actinobacillus pleuropneumoniae isolates according to the antimicrobials to which they showed resistance. Blue colors illustrate the groups with resistance to drugs in fewer than two antimicrobial categories (28.6%), whereas the populations with resistance to drugs in more than three antimicrobial categories are shown in red (71.4%). Numbers indicate the counts of antimicrobial categories to which in the A. pleuropneumoniae isolates showed resistance.
Table 3. The most common antimicrobial resistance patterns (above three isolates) in 133 Actinobacillus pleuropneumoniae isolates.
Sequences of A. pleuropneumoniae in Taiwan
Among these 133 samples, we comprehensively evaluated clinical findings, such as a relatively high mortality rate, a more severe pathological finding, or persistent drug resistance in the clinic. Then a total of 38 A. pleuropneumoniae isolates were selected for NGS analysis. Diversified serotypes of A. pleuropneumoniae can be categorized by phylogeny based on the whole genome. Therefore, the selected 38 A. pleuropneumoniae variants were characterized, and 8 reference sequences were incorporated in this study to generate a phylogenetic tree. Phylogenetic categorization of A. pleuropneumoniae sequences indicated that the bacteria could be separated into five genogroups: serovar 1, serovar 2, serovar 5, serovar 7, and serovar 15 (). In the investigated A. pleuropneumoniae isolates (n = 38), serovar 15 predominated (n = 18, 47.4%) over other serotypes. A total of 7 strains (18.4%) belonged to serotype 7, 6 (15.8%) strains to serotype 5, and 5 strains (13.2%) to serovar 1. Only 2 strains (5.3%) were serotype 2. Taken together, these results indicate that A. pleuropneumoniae serotype 15 is the predominant strain in Taiwan. Strains with high (serotype 1 and 5) and low (serotype 2 and 7) virulence are similarly distributed in Taiwan.
Figure 3. Phylogenetic analysis based on the whole-genome sequences of Actinobacillus pleuropneumoniae strains. A. pleuropneumoniae serovar 1 strain S4074, serovar 1 strain KL16, serovar 2 strain S1536, serovar 2 strain P1875, serovar 5a strain L20, serovar 5b strain K17, serovar 7 strain AP76, serovar 7 strain WF83, and serovar 15 strain HS143 served as clade controls. The circles represent the A. pleuropneumoniae strains isolated from 2017 to 2022. Strains of A. pleuropneumoniae serovars 1, 2, 5, 7, and 15 are labeled orange, green, red, blue, and black, respectively. The scale bars indicate nucleotide substitutions per site.
Resistomes identified in A. pleuropneumoniae isolates
Based on the whole genome of A. pleuropneumoniae, the diversified serotypes and serovar prevalence in Taiwan were shown. We then further analyzed the total genomes of these 38 A. pleuropneumoniae, hoping to disclose possible AMR mechanisms. We identified several ARGs and various antimicrobial resistance profiles (). Functional analysis suggested that these ARGs might contribute resistance to different antimicrobials, as described below. The aminoglycoside resistance genes included aac(6′)-aph(2′’), aadA1, aadA24, aph(3′)-Ia, aph(3′’)-Ib, aph(6)-Id, anti(2′’)-Ia, and ant(6)-Ia. The tet(B) gene is the most commonly occurring tetracycline resistance gene in A. pleuropneumoniae, followed by tet(H), tet(O), and tet(W). The ARGs erm(T) and erm(X) were predicted to lead to macrolide resistance; blaCARB-2 and blaROB-1 were considered beta-lactam resistance genes. The presence of cat and floR genes might result in phenicol resistance. The A. pleuropneumoniae isolates with the sul(2) gene represented sulfonamide-resistant strains. Taken together, the NGS-based resistome results revealed multidrug resistance genes to be predominant (81.6%) (n = 31), followed by tet(B) at 7.9% (n = 3) and floR-tet(B) at 5.3% (n = 2), and no resistance genes were identified in 5.3% (n = 2) of the resistomes. Notably, a plasmid replicon, repUS47, was identified in 13.2% (n = 5) of the isolates. A total of 19 isolates harbored mobile genetic elements (MGEs). Two types of MGEs were identified, ISVAp11 (n = 1) and ISVsa5 (IS10R, n = 18). These results indicated that severe MDR of A. pleuropneumoniae occurred. Furthermore, given the identification of plasmid replicons and/or MGEs, horizontal transfer between A. pleuropneumoniae also poses serious threats to disease control.
Table 4. Antimicrobial resistance genes, plasmid replicons, and MGEs in 38 Actinobacillus pleuropneumoniae isolates identified using next-generation sequencing.
Isolates from the same farms over the years in the NGS analysis were evaluated. The detailed information, including collected data, farm type, and farm size of tested isolates was summarized in Table S2. The 38 A. pleuropneumoniae isolates were collected from 29 different farms. The serial tests of A. pleuropneumoniae isolates from the same farms were found in four different farms, namely, farm numbers 2, 4, 6, and 26. Farms 2 and 4 had four genotypic results within four and three years, respectively, and the serotype affiliation showed a consistent result of serovar 15. Farm 6 has two repeated results. Notably, the first-time isolated A. pleuropneumoniae was serovar 5 (18 June 2020) but the serotype switched to serovar 15 on 28 September 2022. Farm 26 has three repeated results (11 November 2019, 10 March 2020, and 3 November 2020). The serotype affiliations belonged to serovar 7. These findings showed that although the same serovar existed on the same farm for a long period, the alterations of serovars would still occur within the time progression.
Comparison of phenotypic and genotypic susceptibility to antimicrobials
The aforementioned findings showed several ARGs and diversified antimicrobial resistance gene profiles. illustrates the predicted antimicrobials from these corresponding ARGs that might contribute to drug resistance. Compared with phenotypic and genotypic results, the cases of inconsistency of antimicrobials with AMR are labeled in bold. The phenotypic results were in line with the genotyping results in most A. pleuropneumoniae isolates. However, sulfamethoxazole usually was not identified in phenotyping, whereas the NGS results predicted sul(2) gene resistance in these isolates. In the phenotypic results, although enrofloxacin and three cephalosporins (cephalexin, cephalothin, and ceftiofur) seldom showed AMR, the NGS results did not reveal these antimicrobials in all the A. pleuropneumoniae isolates.
Table 5. Comparisons of phenotypes and genotypes of Actinobacillus pleuropneumoniae isolates.
To clarify the concordance of agar dilution methods and NGS, we then analyzed the antimicrobial resistance results of phenotypes and genotypes from all A. pleuropneumoniae isolates (). Substantial to perfect consistency of phenotypic and genotypic susceptibility was found for florfenicol (κ = 0.68), tilmicosin (κ = 0.75), doxycycline (κ = 0.80), and oxytetracycline (κ = 0.83). Moderate phenotype–genotype concordance of kanamycin (κ = 0.43), gentamicin (κ = 0.50), amoxicillin (κ = 0.57), and ampicillin (κ = 0.57) was observed, revealing agreement between the agar dilution methods and NGS results. However, discordance was found for trimethoprim-sulfamethoxazole (κ = 0.14) and streptomycin (κ = 0.19). The kappa values for enrofloxacin and three cephalosporins (cephalexin, cephalothin, and ceftiofur) were zero, which might result from the underdeveloped NGS database.
Table 6. Kappa statistics (agreement) between genotypes and phenotypes of antimicrobial resistance.
Discussion
AMR in clinically relevant bacteria poses considerable threats to public health globally. This study reported the prevalence and AMR patterns of A. pleuropneumoniae in Taiwan. Genetic features were also characterized in 38 A. pleuropneumoniae isolates. Cephalosporins will be the most effective antibacterial agents, whereas resistance levels to tetracycline, aminoglycosides, and beta-lactams are higher. The current study also suggested that MDR became a serious issue and occurred in most A. pleuropneumoniae isolates. Furthermore, genetic analysis, including analysis of ARGs, plasmid replicons, and ISs, revealed the high transmissibility of A. pleuropneumoniae, which also potentially served as targets for disease control and medical intervention.
To date, 19 serovars of A. pleuropneumoniae have been recognized (Stringer et al. Citation2021). In the current study, we identified five serovars, namely, serovars 1, 2, 5, 7, and 15. Among these subtypes, serovars 1 and 5 are generally considered highly virulent strains whereas serovars 2 and 7 are attenuated strains (Chen et al. Citation2022). However, the current study found that serovar 15 is predominant (44.7%), followed by highly virulent (serovar 1 and 5, 31.6%) and attenuated strains (serovar 2 and 7, 23.7%). We speculated that some factors, such as high virulence, viral co-infection or husbandry and management issues enhance the prevalence of serovar 15. A. pleuropneumoniae can be categorized by the different expressions of exotoxins (Stringer et al. Citation2021). The ApxI-containing serovars are more virulent because of their high hemolysis and cell toxicity. Serovars carrying ApxIII and ApxII, such as serovar 15, are also considered virulent, whereas serovars carrying only one toxin gene besides ApxIV show low virulence (Gottschalk and Segura Citation2012; Kardos et al. Citation2022). Furthermore, a serovar 15-like strain isolated in Japan and the HS143 reference strain (serovar 15) were more virulent than other ApxIII-bearing serovars (Koyama et al. Citation2007). Therefore, we presumed that these reasons might explain why serovar 15 is likely to cause epizootics in Taiwan in recent five years. A previous study also showed that A. pleuropneumoniae-infected pigs were usually coinfected with porcine circovirus 2 (PCV2, 56%), Streptococcus suis (24.8%), or the porcine reproductive and respiratory syndrome virus (PRRSV, 23.3%) (Hennig-Pauka et al. Citation2021). The increased frequency of A. pleuropneumoniae coincided with the PCV2 and PRRSV was found in the Hungarian porcine industry (Kardos et al. Citation2022). In Taiwan, we also observed similar findings, in which pigs coinfected with PRRSV and PCV2 were predominant. Therefore, to effectively prevent A. pleuropneumoniae serovar 15 outbreaks in the future, preventive measures that address coinfecting agents should be also taken into consideration.
Several studies have investigated the use of genome sequencing as an alternative to determine AMR in bacteria (Walker et al. Citation2015; Zhao et al. Citation2016); however, it is necessary to validate the correlations between genetic and phenotypic results. In this study, we investigated 38 clinical isolates of A. pleuropneumoniae and found a high correlation between the presence of specific ARGs and AST results for the corresponding antimicrobial agents. However, the results for some antimicrobials are inconclusive and these difficulties might restrict the clinical utility of an NGS approach in certain drugs. The databases for enrofloxacin and several cephalosporins in ResFinders have not been established or are underdeveloped. Thus, we hypothesize that NGS failed to appropriately predict the antimicrobial resistance patterns for these medicines. Furthermore, the most common mechanisms of resistance to fluoroquinolone, which are associated with a mutation in the quinolone-resistance determining regions (QRDRs) within the subunits constituting topoisomerases II (GyrA and GyrB) and IV (ParC and ParE) (Hooper Citation1999) were not detected in all the investigated isolates. The authors hypothesized that few correlations between the antimicrobial mechanisms of A. pleuropneumoniae isolates in this study and the alterations of QRDRs. However, it is more prudent to verify whether the fluoroquinolone-resistant clinical isolates of A. pleuropneumoniae have mutations in the QRDRs. Therefore, we proposed that the traditional ASTs and/or PCR were still needed to determine the antimicrobial patterns in certain veterinary antimicrobial agents. Low concordance has been found in the resistance phenotypes and genotypes of trimethoprim-sulfamethoxazole. These findings might have several explanations. First, commercialized trimethoprim-sulfamethoxazole disks were used in ASTs, and thus, we failed to explain whether the isolates harbored trimethoprim and/or sulfamethoxazole resistance. Second, although we successfully identified the sul2 gene in sulfamethoxazole-resistant strains, there were no trimethoprim resistance genes, such as drfA14 (Bossé et al. Citation2015), identified in the NGS results. These findings might indicate that most of the strains were resistant to sulfamethoxazole, while no trimethoprim resistance occurs at present. Finally, 18 strains (47.4%) exhibited consistent results, including 10 isolates with a negative phenotype (TMP/SMX) and genotype (SMX) and 8 isolates with a positive phenotype and genotype. Notably, 19 strains (50.0%) showed a negative phenotype and a ‘positive’ genotype, which suggested that although resistance to sulfamethoxazole was found, trimethoprim might synergistically enhance the efficacy of sulfamethoxazole. Further investigations, such as the determination of trimethoprim resistance for these isolates or quantitative MIC tests, are highly recommended to address these issues.
AMR poses a considerable threat to public health, and the emergence of AMR is highly correlated with the diversity and mobility of ARGs. Therefore, the characterization of full-length A. pleuropneumoniae genomes is critical for understanding the panresistome, as well as identifying items that affect the transmissibility and pathogenicity of the bacterium. Bacteria can acquire AMR either through mutations in the genome or through horizontal gene transfer (HGT), where HGT of AMR usually involves MGEs, such as plasmids, insertion sequences (ISs), and transposons (Michaelis and Grohmann Citation2023). These results suggest bacterial mobility and thus facilitate rapid spread throughout a bacterial community (Johansson et al. Citation2021). In the current study, we identified a plasmid, repUS47, in five (13.2%) A. pleuropneumoniae isolates. Interestingly, three of the five isolates were isolated from the same husbandry farm during the four years; the remaining two isolates caused relatively high mortality on the farms (compared with our historical data). Furthermore, five isolates with the repUS47 plasmid belonged to serotype 15 based on phylogenetic analysis and serotyping (data not shown). Taken together, these findings could explain why A. pleuropneumoniae isolates with plasmid might result in elevated mortality rates and long-term infections. Previous authors further presumed that although A. pleuropneumoniae serovar 15 is not a traditional strain with high virulence, with the emergence of plasmids, the accumulation and transfer of ARGs increased difficulties in medical intervention (Vrancianu et al. Citation2020). Two ISs, ISVsa5 (IS10R) and ISApl1 (IS30), were identified in half (19 strains) of the isolates. Normally, ISs can transfer or modulate the expression of ARGs, thereby leading to the generation of AMR (Partridge et al. Citation2018). IS10R has been widely reported in the development of colistin resistance (Cannatelli et al. Citation2014) and MDR in Klebsiella pneumoniae in human medicine (Lev et al. Citation2017; Berglund et al. Citation2018). However, few studies have demonstrated the characteristics of IS10R in relation to the change in A. pleuropneumoniae antibiotic susceptibility. Previous studies reported that ISApl1 insertion affected the detection of A. pleuropneumoniae (Tegetmeyer et al. Citation2008) and can interfere with the biosynthesis of certain antigens (To et al. Citation2020). Few previous publications have described the roles of the insertion sequence ISApl1 in AMR. To the best of our knowledge, this is the first study to report that IS10R might influence drug resistance in A. pleuropneumoniae. Further studies are needed to elucidate the detailed mechanisms of whether IS10R, which has caused severe drug resistance in human medicine, participates in the AMR of A. pleuropneumoniae. Co-occurrence between plasmid replicons (repUS47) and ARGs on the same contig was observed in all (5/5) the isolates (Table S3). In these clinical isolates, macrolide resistance gene [erm(T)] co-existed with repUS47 plasmid replicons; to the authors’ best knowledge, few papers reported the correlation between erm(T) co-existed with repUS47. However, a similar association between macrolide resistance gene [erm(B)] and repUS43 and rep9b plasmid replicons was proposed (Amuasi et al. Citation2023). Furthermore, we found that co-occurrence between one MGE (ISVsa5) and ARGs on the same contig was observed in 94.4% (17/18) of the isolates (Table S3). Tetracycline resistance genes [tet(B)] tended to be the most frequently associated with ISVsa5 (n = 14). In two isolates, aminoglycoside resistance genes [aph(3′)-Ia, aph(3′’)-Ib, and aph(6)-Id], beta-lactam resistance gene (blaROB-1), sulfonamide-resistant gene [sul(2)], and tetracycline resistance genes [tet(B)] co-existed with ISVsa5. ARGs encoding resistance to aminoglycoside [aph(3′)-Ia, aph(3′’)-Ib, aph(6)-Id], sulfonamide [sul(2)], and tetracycline [tet(B)] were found in the resulting one. Previous studies also reported similar findings, in which several ARGs tended to occur more often on the same contig with MGEs, such as ISs (Amuasi et al. Citation2023). Nevertheless, few studies have revealed the co-occurrence of plasmid replicons and/or MGEs associated with ARGs encoding resistance to antimicrobials in A. pleuropneumoniae. Therefore, in the present study, genome sequencing not only identified specific ARGs of A. pleuropneumoniae, but also indicated their locations, either within the chromosome or on plasmids (Bossé et al. Citation2017). The identification of plasmid replicon genes and MGEs detected in this study possibly explains the plasticity of A. pleuropneumoniae genomes and supports the potential for horizontal dissemination of the ARGs (Fatoba et al. Citation2022). The dissemination of these genes is intensified by clonal expansion which results when horizontally transferred ARGs become chromosomally integrated (Waddington et al. Citation2022).
The repetition of isolates was evaluated in the current study. The serotype affiliation of Farms 2 and 4 were serovar 15 with several ARGs. Interestingly, in Farm 2, three of the four isolates harbored a plasmid, repUS47, during the four years. In Farm 4, although no plasmids were identified, the NGS analysis identified an IS, ISVsa5, in all the investigated isolates within three years. These results suggest that isolates with MGEs, such as plasmids and IS, might increase the transmissibility and thus result in a long-term infection. Farm 6 has two repeated results. The first-time isolated strain was serovar 5 with an ARG, tet(B). Notably, after two years, the serotype switched to type 15 and six ARGs (aph(3′)-Ia, aph(3′’)-Ib, aph(6)-Id, blaROB-1, sul2, tet(B)) and ISVsa5 were identified. According to these findings, we hypothesized that A. pleuropneumoniae serovar 15 might be the predominant serotype because of the increased transmissibility and enhanced AMR, thereby leading to a long-term infection in Taiwan (Farms 2, 4, and 6). Therefore, although serovar 5 is considered a highly virulent type, serovar 15 gradually posed serious threats in Taiwan. Furthermore, the emergence of MGEs might facilitate HGT, necessitating genetic surveillance in A. pleuropneumoniae. Conversely, no serious mortality nor MGEs have been found in the attenuated strain of serovar 7 (Farm 26), but the aph(3′’)-Ib, blaROB-1, floR, and sul2 genes were repeatedly identified during one year in the same farm. The long-term emergence of MDR also indicated the limited disease control and treatment options for A. pleuropneumoniae in the clinic.
Numerous papers have proposed the possibility of targeting MGEs to combat AMR, as the majority of clinically significant ARGs are located on MGEs (Bossé et al. Citation2017; Johansson et al. Citation2021). One of the categories of MGEs, plasmids, the contributors to the spread of AMR (Bossé et al. Citation2017; Michaelis and Grohmann Citation2023), can be removed by chemical or biological approaches (Vrancianu et al. Citation2020). Therefore, in the current study, NGS results provided extensive information, which potentially revealed the underlying mechanisms that might be covered by traditional ASTs. Furthermore, the determination of antimicrobial susceptibility via phenotyping and genotyping optimized antimicrobial treatment. In the future, based on this platform, a prevention strategy might be established by these genetic markers for the selection of disease-resistant pigs for breeding strategies (Sassu et al. Citation2018). Previous studies reported the whole genome of A. pleuropneumoniae using WGS, which identified several ARGs and/or certain virulence factors. Cohen et al. compared genome sequencing of A. pleuropneumoniae serovar 8 isolates from Norway, Denmark, and the United Kingdom (UK) (Cohen et al. Citation2021). In that study, the authors reported distinct phylogenetic lineages and differences in ARGs in these three regions. Kardos et al. analyzed the genomes of 9 clinical A. pleuropneumonia serovar 13 cases, revealing the low genetic diversity of A. pleuropneumonia serovars in Hungary (Kardos et al. Citation2022). Furthermore, a previous study implemented genome sequencing to obtain 26 A. pleuropneumonia genomes (19 reference serovars and 7 isolates), highlighting their main genetic features and differences (Donà et al. Citation2022). In the present study, all the isolates (38 samples with total genomic profile) were collected from clinical farms, which involved different serotypes that were more commonly observed in Taiwan. We also compared the phenotypic and genotypic results to confirm the AMR patterns in these strains. Furthermore, the prevalences of A. pleuropneumoniae serovars and AMR during the years were also reported. Therefore, the current helps to clarify regional public health issues using WGS for surveillance of AMR in A. pleuropneumoniae. Our research not only provides academic value but also delivers helpful information to the farms.
This study had several limitations. The limited bioinformatic database for veterinary medicine restricted our explorations. The databases for enrofloxacin and cephalosporins have not been established or are underdeveloped. These limitations might impact the overall reliability of NGS. The interactions among identified ARGs remain to be further investigated. Further studies are highly recommended to clarify these issues. Nevertheless, the current study reported the valuable results of antibiotic patterns in A. pleuropneumoniae, which highlighted the need to determine antimicrobial susceptibility to optimize antimicrobial treatment in a case-by-case scenario.
In conclusion, isolates of A. pleuropneumoniae collected in Taiwan from 2017 to 2022 maintained high susceptibility to cephalosporins. However, the increasing resistance against antimicrobials used in pig pleuropneumonia therapy, such as tetracyclines, beta-lactams, and aminoglycosides, remains a tremendous challenge in Taiwan’s porcine industry. The predominant prevalence of A. pleuropneumoniae serovar 15 and the co-infection highlighted the urgent demand to elucidate the pathogenicity of serovar 15. Upcoming studies remain to be performed to assess whether there are differences between A. pleuropneumoniae serovar 15 in their capability to cause disease synergistically with other pathogens of the porcine respiratory diseases. The whole genomes of A. pleuropneumoniae were uncovered by NGS, and the high concordance of phenotypic and genotypic susceptibility to certain antimicrobials highlights the usefulness of this robust diagnostic tool. Furthermore, genome sequencing not only identifies specific ARGs but also provides information on plasmid replicons and/or other MGEs. An accurate AST diagnosis for A. pleuropneumoniae is necessary because it provides the ideal antimicrobial agents for first-line use. However, when the resistant A. pleuropneumoniae harbored certain MGEs, which cannot be detected by the traditional ASTs, the NGS analysis compensates for the limitation. As genomic sequencing of A. pleuropneumoniae, as provided by this study, will likely be of great value to future surveillance and control of this pathogen, both because it increases our comprehensive understanding of AMR patterns, and as it enables the discovery of introductions of new genetic elements (plasmid replicons and MGEs). To unveil the clinical relevance of these genetic characteristics, future studies on pathogenicity within plasmid replicons and/or MGEs are necessary. This study reported and analyzed the phenotypic and genotypic characterization of resistant A. pleuropneumoniae in Taiwan for the first time. When antimicrobial treatment is needed, the principles of prudent use of these agents advise that an accurate diagnosis be made through antimicrobial susceptibility data, and genome results are recommended to support the therapy of choice.
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References
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