Transposition mechanism of ISApl1-the determinant of colistin resistance dissemination.

Multidrug-resistant Enterobacteriaceae, a prominent family of gram-negative pathogenic bacteria, causes a wide range of severe diseases. Strains carrying the mobile colistin resistance (mcr-1) gene show resistance to polymyxin, the last line of defense against multidrug-resistant gram-negative bacteria. However, the transmission of mcr-1 is not well understood. In this study, genomes of mcr-1-positive strains were obtained from the NCBI database, revealing their widespread distribution in China. We also showed that ISApl1, a crucial factor in mcr-1 transmission, is capable of self-transposition. Moreover, the self-cyclization of ISApl1 is mediated by its own encoded transposase. The electrophoretic mobility shift assay experiment validated that the transposase can bind to the inverted repeats (IRs) on both ends, facilitating the cyclization of ISApl1. Through knockout or shortening of IRs at both ends of ISApl1, we demonstrated that the cyclization of ISApl1 is dependent on the sequences of the IRs at both ends. Simultaneously, altering the ATCG content of the bases at both ends of ISApl1 can impact the excision rate by modifying the binding ability between IRs and ISAPL1. Finally, we showed that heat-unstable nucleoid protein (HU) can inhibit ISApl1 transposition by binding to the IRs and preventing ISAPL1 binding and expression. In conclusion, the regulation of ISApl1-self-circling is predominantly controlled by the inverted repeat (IR) sequence and the HU protein. This molecular mechanism deepens our comprehension of mcr-1 dissemination.

A Transposon-Associated CRISPR/Cas9 System Specifically Eliminates both Chromosomal and Plasmid-Borne mcr-1 in Escherichia coli.

The global spread of antimicrobial-resistant bacteria has been one of the most severe threats to public health. The emergence of the mcr-1 gene has posed a considerable threat to antimicrobial medication since it deactivates one last-resort antibiotic, colistin. There have been reports regarding the mobilization of the mcr-1 gene facilitated by ISApl1-formed transposon Tn6330 and mediated rapid dispersion among Enterobacteriaceae species. Here, we developed a CRISPR/Cas9 system flanked by ISApl1 in a suicide plasmid capable of exerting sequence-specific curing against the mcr-1-bearing plasmid and killing the strain with chromosome-borne mcr-1. The constructed ISApl1-carried CRISPR/Cas9 system either restored sensitivity to colistin in strains with plasmid-borne mcr-1 or directly eradicated the bacteria harboring chromosome-borne mcr-1 by introducing an exogenous CRISPR/Cas9 targeting the mcr-1 gene. This method is highly efficient in removing the mcr-1 gene from Escherichia coli, thereby resensitizing these strains to colistin. The further results demonstrated that it conferred the recipient bacteria with immunity against the acquisition of the exogenous mcr-1 containing the plasmid. The data from the current study highlighted the potential of the transposon-associated CRISPR/Cas9 system to serve as a therapeutic approach to control the dissemination of mcr-1 resistance among clinical pathogens.

Transferable Plasmid-Borne mcr-1 in a Colistin-Resistant Shigella flexneri Isolate.

Since the initial discovery of mcr-1 in an Escherichia coli isolate from China, the gene has also been detected in Klebsiella pneumoniae and Salmonella enterica but is rarely reported in other Enterobacteriaceae Here, we report the isolation and identification of a Shigella flexneri strain harboring mcr-1 from stool samples in a pig farm in China from 2009. The MIC of colistin for the isolate is 4 µg/ml. Conjugation assays showed that the donor S. flexneri strain has functional and transferable colistin resistance. Sequencing revealed that mcr-1 was present on a putative composite transposon flanked by inverted repeats of ISApl1IMPORTANCE There are four species of Shigella, and Shigella flexneri is the most frequently isolated species in low- and middle-income countries (LMICs). In this study, we report a functional, transferable, plasmid-mediated mcr-1 gene in S. flexneri We have shown that mcr-1 is located on a novel composite transposon which is flanked by inverted repeats of ISApl1 The host strain is multidrug resistant, and this multidrug resistance is also transferable. The finding of a functional mcr-1 gene in S. flexneri, a human-associated Enterobacteriaceae family member, is a cause for concern as infections due to S. flexneri are the main Shigella infections in most low- and middle-income countries.

In Vitro Study of ISApl1-Mediated Mobilization of the Colistin Resistance Gene mcr-1.

The plasmid-mediated mcr-1 gene encodes a phosphoethanolamine transferase that confers resistance to polymyxins. The mcr-1 gene is associated with insertion sequence ISApl1 (IS30 family). In vitro mobilization assays demonstrated the functionality of the composite transposon structure ISApl1-mcr-1-ISApl1 Transposition generated a 2-bp duplication and occurred in AT-rich DNA regions. This is the first report demonstrating the mobility of the mcr-1 gene by transposition.

Molecular Epidemiology of mcr-1-Positive Polymyxin B-Resistant Escherichia coli Producing Extended-Spectrum ß-Lactamase (ESBL) in a Tertiary Hospital in Shandong, China.

Escherichia coli, a rod-shaped Gram-negative bacterium, is a significant causative agent of severe clinical bacterial infections. This study aimed to analyze the epidemiology of extended-spectrum ß-lactamase (ESBL)-producing mcr-1 -positive E. coli in Shandong, China. We collected 668 non-duplicate ESBL-producing E. coli strains from clinical samples at Shandong Provincial Hospital between January and December 2018, and estimated their minimum inhibitory concentrations (MICs) using a VITEK® 2 compact system and broth microdilution. Next-generation sequencing and bioinformatic analyses identified the mcr-1 gene and other resistance genes in the polymyxin B-resistant strains. The conjugation experiment assessed the horizontal transfer capacity of the mcr-1 gene. Of the strains collected, 24 polymyxin B-resistant strains were isolated with a positivity rate of 3.59% and among the 668 strains, 19 clinical strains carried the mobile colistin resistance gene mcr-1, with a positivity rate of approximately 2.8%. All 19 clinical strains were resistant to ampicillin, cefazolin, ceftriaxone, ciprofloxacin, levofloxacin, and polymyxin B. Seventeen strains successfully transferred the mcr-1 gene into E. coli J53. All transconjugants were resistant to polymyxin B, and carried the drug resistance gene mcr-1. The 19 clinical strains had 14 sequence types (STs), with ST155 (n = 4) being the most common. The whole-genome sequencing results of pECO-POL-29_mcr1 revealed that no ISApl1 insertion sequences were found on either side of the mcr-1 gene. Our study uncovered the molecular epidemiology of mcr-1-carrying ESBL-producing E. coli in the region and suggested horizontal transmission mediated by plasmids as the main mode of mcr-1 transmission.

Global Variation in Escherichia coli mcr-1 Genes and Plasmids from Animal and Human Genomes Following Colistin Usage Restrictions in Livestock.

Antimicrobial resistance (AMR) is a significant global health threat, with multidrug-resistant (MDR) bacterial clones becoming a major concern. Polymyxins, especially colistin, have reemerged as last-resort treatments for MDR Gram-negative infections. However, colistin use in livestock has spread mobile colistin resistance (mcr) genes, notably mcr-1, impacting human health. In consequence, its livestock use was banned in 2017, originating a natural experiment to study bacterial adaptation. The aim of this work was to analyse the changes in the mcr-1 genetic background after colistin restriction across the world. This study analyses 3163 Escherichia coli genomes with the mcr-1 gene from human and livestock hosts, mainly from Asia (n = 2621) and Europe (n = 359). Genetic characterisation identifies IncI2 (40.4%), IncX4 (26.7%), and multidrug-resistant IncHI2 (18.8%) as the most common plasmids carrying mcr-1. There were differences in plasmids between continents, with IncX4 (56.6%) being the most common in Europe, while IncI2 (44.8%) was predominant in Asia. Promoter variants related to reduced fitness costs and ISApl1 showed a distinct pattern of association that appears to be associated with adaptation to colistin restriction, which differed between continents. Thus, after the colistin ban, Europe saw a shift to specialised mcr-1 plasmids as IncX4, while ISApl1 decreased in Asia due to changes in the prevalence of the distinct promoter variants. These analyses illustrate the evolution of mcr-1 adaptation following colistin use restrictions and the need for region-specific strategies against AMR following colistin restrictions.

Intracellular Transposition of Mobile Genetic Elements Associated with the Colistin Resistance Gene mcr-1.

Mobile colistin resistance (mcr) genes are often located on conjugative plasmids, where their association with insertion sequences enables intercellular and intracellular dissemination throughout bacterial replicons and populations. Multiple mcr genes have been discovered in every habitable continent, in many bacterial species, on both plasmids and integrated into the chromosome. Previously, we showed the intercellular transfer of mcr-1 on an IncI1 plasmid, pMCR-E2899, between strains of Escherichia coli. Characterizing the intracellular dynamics of mcr-1 transposition and recombination would further our understanding of how these important genes move through bacterial populations and whether interventions can be put in place to stop their spread. In this study, we aimed to characterize transfer events from the mcr-1-containing transposon Tn7511 (ISApl1-mcr-1-pap2-ISApl1), located on plasmid pMCR-E2899, using the pBACpAK entrapment vector. Following the transformation of pBACpAK into our DH5α-Azir/pMCR-E2899 transconjugant, we captured ISApl1 in pBACpAK multiple times and, for the first time, observed the ISApl1-mediated transfer of the mcr-1 transposon (Tn7511) into the chromosome of E. coli DH5α. Whole-genome sequencing allowed us to determine consensus insertion sites of ISApl1 and Tn7511 in this strain, and comparison of these sites allowed us to explain the transposition events observed. These observations reveal the consequences of ISApl1 transposition within and between multiple replicons of the same cell and show mcr-1 transposition within the cell as part of the novel transposon Tn7511. IMPORTANCE By analyzing the intracellular transfer of clinically relevant transposons, we can understand the dissemination and evolution of drug resistance conferring mobile genetic elements (MGEs) once a plasmid enters a cell following conjugation. This knowledge will help further our understanding of how these important genes move through bacterial populations. Utilizing the pBACpAK entrapment vector has allowed us to determine the mobility of the novel mcr-1-containing transposon Tn7511.

Emergence of the mcr-1 colistin resistance gene in extended-spectrum ß-lactamase-producing Klebsiella pneumoniae in Taiwan.

OBJECTIVES: The resistance ofmcr-1-carrying Enterobacteriaceae to polymyxins, which are last-resort antibiotics, has raised great concern worldwide. In this study, four mcr-1-carrying plasmids isolated from Klebsiella pneumoniae clinical isolates were completely sequenced and the genome composition of the mcr-1-carrying plasmids was analysed. METHODS: Antimicrobial resistance genes and antimicrobial susceptibility of fourmcr-1-carrying K. pneumoniae isolates were characterised. Comparison of mcr-1-carrying plasmids with closely related plasmids and analysis of the mcr-1 gene cassette were performed. RESULTS: The genome composition of the fourmcr-1-carrying plasmids revealed the Tn6330 and Tn6390 cassettes embedded in two IncHI1-type plasmids, ΔTn6330 in an IncHI2-type plasmid, and mcr-1-pap2 in an IncX4-type plasmid. We also predicted the intermediate structures of the Tn6330 and Tn6390 cassettes. CONCLUSION: Dissemination of the colistin resistant genemcr-1 in Taiwan could have been driven by various plasmids and mobile gene cassettes. Evolution of the genetic environment has led to diversity in the mcr-1 gene among plasmids. This work sheds light on the urgent need for continued surveillance of the worldwide distribution of mcr-1 and evaluates the public-health risk of colistin resistance.

Identification of mcr-1 and a novel chloramphenicol resistance gene catT on an integrative and conjugative element in an Actinobacillus strain of swine origin.

The aim of this study was to characterize a mcr-1-carrying integrative and conjugative element (ICE) in a novel Pasteurellaceae-like bacteria of swine origin. The mcr-1-positive GY-402 strain, recovered from a pig fecal sample, was subjected to whole genome sequencing with the combination of Illumina Hiseq and MinION platforms. Genome-based taxonomy revealed that strain GY-402 exhibited highest ANI value (84.89 %) to Actinobacillus succinogenes, which suggested that it represented a novel Actinobacillus species. Sequence analysis revealed that mcr-1 was clustered with eight other resistance genes in the MDR region of a novel ICE element, named ICEAsp1. Inverse PCR and mating assays showed that ICEAsp1 is active and transferrable. In addition, six circular forms mediated by four ISApl1 elements were detected with different inverse PCR sets, indicating that flexible composite transposons could be formed by pairwise combinations of multiple IS copies. Cloning experiment and phylogenetic analysis revealed that the novel Cat protein, designated CatT, belongs to type-A family and confers resistance to chloramphenicol. In conclusion, this is, to the best of our knowledge, the first report of mcr-1 gene on ICE structure and also in Pasteurellaceae bacteria. The diverse composite transposons mediated by multicopy IS elements may facilitate the dissemination of different resistance genes.

Updates on the global dissemination of colistin-resistant Escherichia coli: An emerging threat to public health.

Colistin drug resistance is an emerging public health threat worldwide. The adaptability, existence and spread of colistin drug resistance in multiple reservoirs and ecological environmental settings is significantly increasing the rate of occurrence of multidrug resistant (MDR) bacteria such as Escherichia coli (E. coli). Here, we summarized the reports regarding molecular and biological characterization of mobile colistin resistance gene (mcr)-positive E. coli (MCRPEC), originating from diverse reservoirs, including but not limited to humans, environment, waste water treatment plants, wild, pets, and food producing animals. The MCRPEC revealed the abundance of clinically important resistance genes, which are responsible for MDR profile. A number of plasmid replicon types such as IncI2, IncX4, IncP, IncX, and IncFII with a predominance of IncI2 were facilitating the spread of colistin resistance. This study concludes the distribution of multiple sequence types of E. coli carrying mcr gene variants, which are possible threat to "One Health" perspective. In addition, we have briefly explained the newly known mechanisms of colistin resistance i.e. plasmid-encoded resistance determinant as well as presented the chromosomally-encoded resistance mechanisms. The transposition of ISApl1 into the chromosome and existence of intact Tn6330 are important for transmission and stability for mcr gene. Further, genetic environment of co-localized mcr gene with carbapenem-resistance or extended-spectrum ß-lactamases genes has also been elaborated, which is limiting human beings to choose last resort antibiotics. Finally, environmental health and safety control measures along with spread mechanisms of mcr genes are discussed to avoid further propagation and environmental hazards of colistin resistance.

Characterization of nontypeable Actinobacillus pleuropneumoniae isolates.

Two Actinobacillus pleuropneumoniae isolates from clinical cases of porcine pleuropneumonia in Japan were positive in the capsular serovar 15-specific PCR assay, but nontypeable (NT) in the agar gel precipitation (AGP) test. Nucleotide sequence analysis of gene clusters involved in the biosynthesis of capsular polysaccharide (CPS) and lipopolysaccharide O-polysaccharide (O-PS) revealed that both clusters contained transposable element ISApl1 of A. pleuropneumoniae belonging to the IS30 family. Immunoblot analysis revealed that these 2 isolates could not produce O-PS. We conclude that the ISApl1 of A. pleuropneumoniae can interfere in the biosynthesis of both CPS and O-PS.

Plasmid Dynamics of mcr-1-Positive Salmonella spp. in a General Hospital in China.

Salmonella is an important food pathogen that can cause severe gastroenteritis with more than 600,000 deaths globally every year. Colistin (COL), a last-resort antibiotic, is ineffective in bacteria that carry a functional mcr-1 gene, which is often spread by conjugative plasmids. Our work aimed to understand the prevalence of the mcr-1 gene in clinical isolates of Salmonella, as the frequency of occurrence of the mcr-1 gene is increasing globally. Therefore, we analyzed 689 clinical strains, that were isolated between 2009 and late 2018. The mcr-1 gene was found in six strains, which we analyzed in detail by whole genome sequencing and antibiotic susceptibility testing, while we also provide the clinical information on the patients suffering from an infection. The genomic analysis revealed that five strains had plasmid-encoded mcr-1 gene located in four IncHI2 plasmids and one IncI2 plasmid, while one strain had the chromosomal mcr-1 gene originated from plasmid. Surprisingly, in two strains the mcr-1 genes were inactive due to disruption by insertion sequences (ISs): ISApl1 and ISVsa5. A detailed analysis of the plasmids revealed a multitude of ISs, most commonly IS26. The IS contained genes that meditate broad resistance toward most antibiotics underlining their importance of the mobile elements, also with respect to the spread of the mcr-1 gene. Our study revealed potential reservoirs for the transmission of COL resistance and offers insights into the evolution of the mcr-1 gene in Salmonella.

First report of the multiresistance gene cfr in Pasteurella multocida strains of avian origin from China.

OBJECTIVES: The aim of this study was to investigate the presence and genetic environment of the multiresistance gene cfr gene in Pasteurella multocida of avian origin from China. METHODS: A total of 113 P. multocida isolates were collected from sick poultries (ducks, chickens and geese) from 2003 to 2016 in Southern China and were screened for the presence of the cfr gene by PCR. The cfr-carrying P. multocida strains were subjected to antimicrobial susceptibility testing, S1 nuclease PFGE and Southern blot hybridisation, conjugative transfer and analysis of genetic environment of the cfr gene. RESULTS: Among 113 P. multocida isolates, strains FJ6671 and FJ6683 from Muscovy duck harboured the cfr gene and presented a multiresistant phenotype. The cfr gene in the two strains was located on an ∼40-kb conjugative plasmid in different genetic environments, including ISApl12-cfr-IS26 and IS26-cfr-IS256. CONCLUSIONS: These results demonstrate plasmid-carried cfr in P. multocida and suggest that transposition and homologous recombination mediated by IS26, ISApl1 and IS256 might have played an important role in transfer of the cfr gene in P. multocida. To the best of our knowledge, this is the first report of the cfr gene in P. multocida. Active and ongoing surveillance of cfr in P. multocida is urgently warranted.

Identification of a multiple drug-resistance gene island in the Haemophilus parasuis chromosome.

OBJECTIVES: The aim of this study was to determine the mechanisms and molecular characterisation of one strain (HPS412) of Haemophilus parasuis, which exhibited high MICs of antimicrobial susceptibility. METHODS: A total of 113 H. parasuis strains isolated from pigs suffering from polyserositis, pneumonia or meningitis in China and screened them for antimicrobial susceptibility. Susceptibility testing of the minimal inhibitory concentrations (MIC) was determined in fastidious medium consisting of tryptone soya broth (TSB) containing 5% bovine serum and 10µg/mL NAD in 96-well microtiter plates. The genomic DNA was completely sequenced by combining PacBio RS II and Illumina HiSeq 4000 platforms. Gene prediction was performed using Glimmer v.3.02 with Hidden Markov models. RESULTS: One strain (HPS412) exhibited high MICs of sulfamethoxazole (256µg/mL), tetracycline (128µg/mL), streptomycin (128µg/mL), gentamicin (128µg/mL), amoxicillin (128µg/mL), chloramphenicol (64µg/mL), penicillin (64µg/mL) and cefaclor (64µg/mL). Sequence analysis showed that numerous drug-resistance genes including tet(B), blaROB-1, sul2, catIII, aph(3″)-Ib, aph(6)-Id and aph(3')-Ia were present in a chromosomal gene island as adjacent duplicate copies and the rep-orf3-blaROB-1 structure most likely had a direct plasmid origin. The tet(B) and blaROB-1 were flanked on one or both by ISApl1 elements. CONCLUSIONS: The acquisition of blaROB-1 and the other antibiotic resistance genes was related to the presence of ISApl1. ISApl1 plays important roles in the horizontal transmission of antibiotic resistance genes.

The ISApl1 2 Dimer Circular Intermediate Participates in mcr-1 Transposition.

Objectives: The mobile colistin resistance gene mcr-1 is a serious threat to global human and animal health. The composite transposon Tn6330 and its circular intermediate were proposed to be involved in the spread of mcr-1 but their roles remain poorly understood. Methods: To further explore the intermediates during the transposition of Tn6330, we engineered Escherichia coli strains that carry an intact Tn6330 transposon or its deletion derivatives. PCR assays were performed to detect IR-IR junctions and possible circular intermediates. We carried out transposition experiments to calculate transposition frequency. The transposition sites were characterized by whole genome sequence and ISMapper-based analyses. Results: The presence of an intact Tn6330 was demonstrated to be essential for the successful transposition of mcr-1, although both Tn6330 and Tn6330-ΔIR could form circular intermediates. The insertion sequence junction structure was observed in all constructed plasmids but the ISApl1 dimer was only formed in one construct containing an intact Tn6330. The average frequency of mcr-1 transposition in an E. coli strain possessing an intact Tn6330 was ∼10-6 per transformed cell. We identified 27 integration sites for the Tn6330 transposition event. All the transposition sites were flanked by 2 bp target duplications and preferentially occurred in AT-rich regions. Conclusion: These results indicate that mcr-1 transposition relies on the presence of an intact Tn6330. In addition, formation of the tandem repeat ISApl1 2 could represent a crucial intermediate. Taken together, the current investigations provide mechanistic insights in the transposition of mcr-1.

Key features of mcr-1-bearing plasmids from Escherichia coli isolated from humans and food.

BACKGROUND: Mcr-1-harboring Enterobacteriaceae are reported worldwide since their first discovery in 2015. However, a limited number of studies are available that compared full-length plasmid sequences of human and animal origins. METHODS: In this study, mcr-1-bearing plasmids from seven Escherichia coli isolates recovered from patients (n = 3), poultry meat (n = 2) and turkey meat (n = 2) in Switzerland were further analyzed and compared. Isolates were characterized by multilocus sequence typing (MLST). The mcr-1-bearing plasmids were transferred by transformation into reference strain E. coli DH5α and MCR-1-producing transformants were selected on LB-agar supplemented with 2 mg/L colistin. Purified plasmids were then sequenced and compared. RESULTS: MLST revealed six distinct STs, illustrating the high clonal diversity among mcr-1-positive E. coli isolates of different origins. Two different mcr-1-positive plasmids were identified from a single E. coli ST48 human isolate. All other isolates possessed a single mcr-1 harboring plasmid. Transferable IncI2 (size ca. 60-61 kb) and IncX4 (size ca. 33-35 kb) type plasmids each bearing mcr-1 were found associated with human and food isolates. None of the mcr-1-positive IncI2 and IncX4 plasmids possessed any additional resistance determinants. Surprisingly, all but one of the sequenced mcr-1-positive plasmids lacked the ISApl1 element, which is a key element mediating acquisition of mcr-1 into various plasmid backbones. CONCLUSIONS: There is strong evidence that the food chain may be an important transmission route for mcr-1-bearing plasmids. Our data suggest that some "epidemic" plasmids rather than specific E. coli clones might be responsible for the spread of the mcr-1 gene along the food chain.

Study of mcr-1 Gene-Mediated Colistin Resistance in Enterobacteriaceae Isolated from Humans and Animals in Different Countries.

In this study, we aim to characterize the genetic environment of the plasmid-mediated colistin resistance gene mcr-1 in 25 Escherichia coli and seven Klebsiella pneumoniae strains from different countries and continents. Multilocus sequence typing, conjugation experiments, plasmid typing, and the presence and location of the insertion sequence ISApl1 were investigated. Whole genome sequencing of four E. coli was performed to analyse the genetic environment of the mcr-1 gene. Colistin minimum inhibitory concentration of mcr-1 strains varied from 3 to 32 µg/mL. Six E. coli sequence types were detected: ST 4015, ST 3997, ST 10, ST 93, ST 48, and ST 648. IncHI2, IncI2, and IncP plasmid types were predominant and were unrelated to a specific country of origin. ISApl1 was found in 69% of analysed plasmids that were mainly around the mcr-1 gene. Analysis of four closed mcr-1 plasmids revealed the integration of mcr-1 into hotspots. We found that the spread of mcr-1 gene was due to the diffusion of a composite transposon and not to the diffusion of a specific plasmid or a specific bacterial clone. The ease with which the mcr-1 gene integrates into various regions facilitates its dissemination among bacteria and explains its large diffusion all over the world, both in animals and in humans.

Nucleotide sequence analysis of a DNA region involved in capsular polysaccharide biosynthesis reveals the molecular basis of the nontypeability of two Actinobacillus pleuropneumoniae isolates.

The aim of our study was to reveal the molecular basis of the serologic nontypeability of 2 Actinobacillus pleuropneumoniae field isolates. Nine field strains of A. pleuropneumoniae, the causative agent of porcine pleuropneumonia, were isolated from pigs raised on the same farm and sent to our diagnostic laboratory for serotyping. Seven of the 9 strains were identified as serovar 15 strains by immunodiffusion tests. However, 2 strains, designated FH24-2 and FH24-5, could not be serotyped with antiserum prepared against serovars 1-15. Strain FH24-5 showed positive results in 2 serovar 15-specific PCR tests, whereas strain FH24-2 was only positive in 1 of the 2 PCR tests. The nucleotide sequence analysis of gene clusters involved in capsular polysaccharide biosynthesis of the 2 nontypeable strains revealed that both had been rendered nontypeable by the action of ISApl1, a transposable element of A. pleuropneumoniae belonging to the IS30 family. The results showed that ISApl1 of A. pleuropneumoniae can interfere with both the serologic and molecular typing methods, and that nucleotide sequence analysis across the capsular gene clusters is the best means of determining the cause of serologic nontypeability in A. pleuropneumoniae.