Copies Of IS6110 Research Articles

Coexistence of three blaKPC-2 genes on an IncF/IncR plasmid in ST11 Klebsiella pneumoniae.

Here we report the finding of three copies of the blaKPC-2 gene on a plasmid in ST11 Klebsiella pneumoniae. Carbapenem-resistant K. pneumoniae clinical strain WCHKP2 was subjected to whole-genome sequencing (WGS) using both a short-read Illumina HiSeq X10 platform and long-read MinION sequencer. Hybrid assembly was performed using Unicycler, and contigs were corrected using Pilon. Based on WGS, the sequence type (ST), capsular type, plasmid replicon type and plasmid multilocus sequence type were determined and virulence and antimicrobial resistance genes were identified. Mating was performed to identify a self-transmissible plasmid mediating carbapenem resistance. Strain WCHKP2 was resistant to imipenem [minimum inhibitory concentration (MIC)=64μg/mL] and meropenem (MIC=128μg/mL). Strain WCHKP2 had a 5477148-bp circular chromosome, two small ColRNAI-like plasmids (5596bp and 10060bp), and one large plasmid (177516bp, designated pKPC2_020002) containing an IncR and an IncFII replicon. Surprisingly, there were three copies of the blaKPC-2 carbapenemase gene on pKPC2_020002, which was not self-transmissible. Each of the blaKPC-2 genes was located in the same genetic context with insertion sequence ISKpn27 upstream and ISKpn6 downstream and bracketed by IS26. The three copies of the IS26-ISKpn27-blaKPC-2-ISKpn6-IS26 unit were present in tandem. Here we report the surprising co-existence of three copies of blaKPC-2 on an IncR/IncF plasmid due to the action of IS26. Multiple copies of IS26 are a key factor generating genetic plasticity and could mediate the multiplication of resistance genes.

Combined testing for herpes simplex virus and Mycobacterium tuberculosis DNA in cerebrospinal fluid of patients with aseptic meningitis in Burkina Faso, West Africa.

Little is known about the involvement of herpes simplex virus (HSV) or Mycobacterium tuberculosis (MTB) as potentially curable causes of central nervous system (CNS) infections in sub-Saharan Africa. In this study, we developed a PCR assay dedicated to simultaneous testing of HSV1/HSV2 and MTB in Burkina Faso, a country where HSV is neglected as a cause of CNS infection and where TB prevalence is high. A consensus HSV1/HSV2 set of primers and probe were designed and combined to primers and probe targeting the IS6110 repetitive insertion sequence of MTB. Analytical performances of the assay were evaluated on reference materials. Cerebrospinal fluid (CSF) collected from subjects with aseptic meningitis was tested for HSV1/HSV2 and MTB DNA. The UL29 gene was chosen as a highly conserved region targeted by the HSV1/HSV2 nucleic acid test. The lower limits of detection were estimated to be 2.45copies/µL for HSV1, 1.72copies/µL for HSV2, and 2.54 IS6110 copies per µL for MTB. The PCR was used in 202 CSF collected from subjects suspected of aseptic meningitis. Five samples (2.46%) tested positive, including two children positive for HSV1 (0.99%) and three adults tested positive for MTB (1.47%). Using an in-house real-time PCR assay, we showed that both HSV and MTB are etiologic pathogens contributing to aseptic meningitis in Burkina Faso. This molecular test may have clinical utility for early diagnosis for those treatable CNS infections.

Carriage of blaKPC-2 by a virulence plasmid in hypervirulent Klebsiella pneumoniae.

To characterize a plasmid in a K1 hypervirulent Klebsiella pneumoniae (HvKP) strain encoding both hypervirulence and carbapenem resistance phenotypes. Plasmids from HvKP strain KP70-2 were subjected to whole-plasmid sequencing using both the Illumina NextSeq 500 sequencing platform and Nanopore MinION sequencer platforms. A hybrid virulence- and resistance-encoding plasmid of 240 kb, harbouring both the virulence gene rmpA2 and the carbapenemase gene blaKPC-2, was recovered from a clinical HvKP strain. Designated pKP70-2, the plasmid was found to be almost structurally identical to various known hypervirulence-encoding plasmids harboured by other HvKP strains, except for an extra MDR-encoding region located within the genetically conserved plasmid backbone. This MDR region was flanked by two copies of IS26 in the same orientation, one at each end and linked to an external 8 bp (CTAAAATT) product of target site duplications, suggesting that an insertion event was responsible for the integration of the MDR region into the virulence plasmid. The MDR region was also found to harbour mobile elements that in turn contain the antibiotic resistance genes dfrA14 and blaKPC-2. Based on the genetic composition of pKP70-2, we postulate that the multiple insertion elements that it harbours were responsible for mediating the plasmid recombination events that underlie continuous emergence and genetic adaptation of novel resistance- and virulence-encoding mobile elements in K. pneumoniae.

Coexistence of Two blaNDM-5 Genes on an IncF Plasmid as Revealed by Nanopore Sequencing.

In a carbapenem-resistant Escherichia coli clinical isolate of sequence type 167, two copies of blaNDM-5 were found on a 144,225-bp IncF self-transmissible plasmid of the F36:A4:B- type. Both blaNDM-5 genes were located in 11,065-bp regions flanked by two copies of IS26 The two regions were identical in sequence but were present at different locations on the plasmid, suggesting a duplication of the same region. This study highlights the complex genetic contexts of blaNDM-5.

Cryptic transmission of ST405 Escherichia coli carrying blaNDM-4 in hospital

Three carbapenem-resistant Escherichia coli were recovered from rectal swabs of different patients in a tertiary hospital and were found carrying blaNDM-4, an uncommon blaNDM variant. Genome sequences of the isolates were obtained using Illumina technology and the long-read MinION sequencer. The isolates belonged to ST405 and phylogenetic group D, a globally distributed lineage associated with antimicrobial resistance. In addition to blaNDM-4, the three isolates carried 14 known resistance genes including the extended-spectrum β-lactamase gene blaCTX-M-15. There were only 1 or 2 SNPs between the isolates, suggesting a common origin and cryptic transmission in hospital. blaNDM-4 was located on a 46.5-kb IncFIA self-transmissible plasmid, which may facilitate further dissemination of blaNDM-4. Two copies of IS26 bracketed a 14.6-kb region containing blaNDM-4 and have the potential to form a composite transposon for mediating the mobilization of blaNDM-4.

Comparative genomics of rmtB-carrying IncI1 ST136 plasmids in avian escherichia coli isolates from chickens in China

Eight rmtB-carrying avian Escherichia coli strains from a farm in China were characterised in our previous study, but little is known about the backbones and entire multiresistance regions (MRRs) of these plasmids. Here, three rmtB-carrying IncI1 ST136 plasmids were analysed by whole-plasmid sequencing and were compared. These plasmids were composed of an 83 470-bp IncI1 backbone carrying genes responsible for plasmid replication, transfer, maintenance and stability functions, as well as a 17 330-bp MRR for pEC006 and pEC007, and a 34 626-bp MRR for pEC008. Plasmid pEC006 was not transferable, thus truncation of the traI gene may explain the inability to conjugate. pEC008 harboured the blaTEM-1, rmtB, aacC2, tetA, floR and strAB genes as well as a class 1 integron cassette array (|dfrA12|orfF|aadA2|), which were interspersed with different mobile elements, including Tn2, Tn1721, Tn1722, Tn5393, ISCfr1, IS5057, ISCR1 and ISCR2, and three copies of IS26. The MRR of pEC008 may have resulted from transposition of Tn1722 into the plasmid backbone. Acquisition and rearrangement of MRRs demonstrated the accumulation of different resistance determinants.

Complete sequence of pBM413, a novel multidrug resistance megaplasmid carrying qnrVC6 and blaIMP-45 from pseudomonas aeruginosa

This study aimed to characterise a novel multidrug resistance megaplasmid carrying qnrVC6 and blaIMP-45 from Pseudomonas aeruginosa strain Guangzhou-Pae617 isolated from a patient hospitalised in Guangzhou, China, in 2012. The plasmid pBM413 has a length of 423 017 bp and an average G + C content of 56.41%. A qnrVC6 gene flanked by two copies of insertion sequence (IS) elements ISCR1, a multiresistance class 1 integron In786 containing aacA4–blaIMP-45–blaOXA-1–catB3 cassettes, an armA gene, and an aphA7 gene flanked by two copies of IS26 were identified. To our knowledge, this is the first identification of a qnrVC6 gene in P. aeruginosa.

RFLP clusters of rifampicin resistant and susceptible Mycobacterium tuberculosis strains in Western province of Sri Lanka.

Continuous studies on genetic diversity of Mycobacterium tuberculosis could enhance the awareness on transmission, control and prevention of tuberculosis (TB). In this study, we investigated current genetic diversity of TB and rifampicin resistant TB by, Restriction Fragment Length Polymorphism (RFLP) based on fingerprinting of the IS6110 insertion sequence, in the Western province of Sri Lanka, the famous touristic destination with the highest TB burden in the country. Genomic DNA extracted from susceptible and rifampicin resistant TB strains (confirmed for rpoB gene point mutations) were digested with PvuII restriction enzyme, electrophoresed and subjected to Southern transfer. The blots were hybridised with IS6110 probe and visualized using a chemiluminescence detection. The number of copies of IS6110 per isolate varied from 1 to 14. The dendrogram revealed a total of 68 distinct strains among 77 TB isolates and they belonged to nine clusters. Both rifampicin resistant and susceptible strains were distributed in all clusters. This evaluation revealed the absence of genetically identical or strong relatedness between susceptible and resistant isolates. However, clonal expansion was detected in transmission of both TB and rifampicin resistant TB. In addition, the resistant isolates having the novel mutation had no clonal relatedness. This is the first observational study regarding clonal expansion of TB in Sri Lanka. Thus, further investigation on genotypes, clonal expansion and transmission of drug resistance using additional markers would be useful for controlling TB.

Targeted conservative formation of cointegrates between two DNA molecules containing IS26 occurs via strand exchange at either IS end.

We recently proposed a model for targeted, conservative cointegrate formation between DNA molecules each containing a copy of IS26, that involves Tnp26-catalyzed strand exchange occurring at either the two left ends or the two right ends of the IS. Here, this model was validated by altering the bases at the outer left terminus, right terminus or both termini of one IS26. The correct bases at both ends were required in the untargeted replicative mode. However, when only one end was altered in one participating IS the frequency of targeted, conservative cointegrate formation was not reduced. The distribution of the altered bases in the cointegrates confirmed that the reaction occurred at the end where the terminal bases of both IS were correct, and cointegrates were not formed when both ends of the same IS were altered. The terminal bases of the active IS26 were also required to support deletion of the aphA1a translocatable unit (TU) from Tn4352B. The choices made by an incoming TU with a wild-type IS26 when the target plasmid included one wild-type IS26 and one with a frameshift in tnp26 demonstrated that Tnp26 exhibits a strong preference for cis action.

The role of IS26 in evolution of a derivative of the virulence plasmid of Salmonella enterica serovar Enteritidis which confers multiple drug resistance

pUO-SeVR1 is a resistance derivative of pSEV, the virulence plasmid specific of Salmonella enterica serovar Enteritidis. It was first detected in a Spanish isolate involved in gastroenteritis, but closely related plasmids are widespread in highly invasive isolates originating from Africa. According to its nucleotide sequence, pUO-SeVR1 consists of 110,982bp with a GC content of 53.2%. It confers resistance to multiple antimicrobial agents and contains a wealth of mobile genetic elements, including 14 copies of IS26 which have played a major role in the evolution of the plasmid. An intact copy of IS4321 and remnants of many other transposable elements (Tn2, Tn21, Tn1721 and Tn5393) are also detected. Most of the pSEV sequence is conserved in pUO-SeVR1 but small deletions, major inversions and the insertion of two foreign regions (of 38.7 and 13.6kb, where the resistance genes are clustered), have occurred. Plasmids of the IncL/M, IncN and IncQ groups could have contributed to the assembly of the resistance regions.

Characterization of Two Multidrug-Resistant IncA/C Plasmids from the 1960s by Using the MinION Sequencer Device.

Two A/C incompatibility group (IncA/C family) plasmids from the 1960s have been sequenced and classified into the A/C2 type 1 group. R16a and IP40a contain novel antibiotic resistance islands and a complete GIsul2 genomic island not previously found in the family. In the 173.1-kb R16a, the 29.9-kb antibiotic resistance island (ARI) is located in a unique backbone position not utilized by ARIs. ARIR16a consists of Tn1, Tn6020, and Tn6333, harboring the resistance genes blaTEM-1D and aphA1b and a mer module, respectively; a truncated Tn5393 copy; and a gene cluster with unknown function. Plasmid IP40a is 170.4 kb in size and contains a 5.6-kb ARI inserted into the kfrA gene. ARIIP40a carrying blaTEM-1D and aphA1b genes is composed of Tn1 with a Tn6023 insertion. Additionally, IP40a harbors single IS2, IS186, and Tn1000 insertions scattered in the backbone; an IS150 copy in GIsul2; and a complete Tn6333 carrying a mer module at the position of ARIR16a Loss of resistance markers in R16a, IP40a, and R55 was observed during stability tests. Every phenotypic change proved to be the result of recombination events involving mobile elements. Intramolecular transposition of IS copies that generated IP40a derivatives lacking large parts of the backbone could account for the formation of other family members, too. The MinION platform proved to be a valuable tool in bacterial genome sequencing since it generates long reads that span repetitive elements and facilitates full-length plasmid or chromosome assembly. Nanopore technology enables rapid characterization of large, low-copy-number plasmids and their rearrangement products.

A Prototype Biomarker Detector Combining Biomarker Extraction and Fixed Temperature PCR.

PCR is the most sensitive molecular diagnostic available for infectious diseases. The goal for low-resource settings is a simple, inexpensive instrument. Toward this goal, we previously published a self-contained sample preparation instrument that uses magnetics and prearrayed reagents in thin tubing to extract nucleic acids and perform isothermal amplification and detection of extracted biomarkers. To incorporate PCR thermal cycling, after biomarker is magnetically extracted from a patient sample, the section of tubing containing the extracted biomarker and PCR reagents is alternately positioned within two constant temperature blocks. This instrument was evaluated initially by extracting and amplifying a 140 bp fragment of the IS6110 sequence of tuberculosis from TE buffer. The mean cycle threshold for 5 × 10(6) copies of IS6110 was 25.5 ± 1.5 cycles (n = 4), which was significantly different from negative control samples (34.0 ± 2.6 cycles; n = 3). Using a more clinically relevant sample, we extracted and amplified Plasmodium falciparum DNA from malaria-infected human blood cultures. The average cycle threshold for 1% parasitemia samples was 24.7 ± 1.5 cycles (n = 3) and significantly different from negatives (31.5 ± 2.1 cycles; n = 3). This approach integrates biomarker extraction, PCR amplification, and detection in a simple, linear tubing design with potential for use as a low-resource instrument.

Plasmid mediated fosfomycin resistance and its mechanism in extended-spectrum β-lactamases producing Klebsiella pneumonia

Objective To investigate the fosfomycin resistance and its mechanism in extended-spectrum β-lactamases (ESBL) producing Klebsiellapneumoniae (K. pneumoniae) from clinical samples. Methods Seventy-nine isolates of ESBL-producing clinical K. pneumoniae were collected at Sir Run Run Shaw Hospital, Zhenjiang University School of Medicine from July 2014 to December 2014. Minimal inhibitory concentration (MIC) of fosfomycin was determined by the agar dilution method, and the susceptibility breakpoint was interpreted as ≤32 mg/L according to European Committee on Antimicrobial Susceptibility Testing (EUCAST). Phylogenetic clonal patterns were analyzed by pulse-field gel electrophoresis (PFGE), and plasmid size and similarity were revealed by nuclease assay. Filter membrane conjugation assay was carried out for determination of resistance transfer by plasmid. All the isolates were screened for plasmid-mediated fosfomycin resistance genes by polymerase chain reaction (PCR) amplification, and the structures of up- and down-stream resistance genes were analyzed. Results Nine of 79 isolates of ESBL-producers were resistant against fosfomycin, and the resistance rate was 11.4%. PFGE and S1-PFGE revealed that all the 9 fosA3-harboring isolates were not clone related and not plasmid similar. All of the 9 resistant isolates were fosA3 positive, which was one of the plasmid mediated fosfomycin resistance genes. No other fosA or fosC2-positive resistant genes were found. fosA3 gene coded by resistant isolates was found to be flanked by two copies of IS26. Conclusions Fosfomycin resistant isolates are emerged in ESBL-producing clinical K. pneumoniae. Producing fosfomycin-modifying enzyme fosA3 coded by plasmid is the major mechanism in fosfomycin resistant isolates. The IS26 element accelerates the resistance genes dissemination. Key words: Klebsiella pneumoniae; Fosfomycin; Resistance; Plasmids

Evolutionary Trends of the Transposase-Encoding Open Reading Frames A and B (orfA and orfB) of the Mycobacterial IS6110 Insertion Sequence.

BackgroundThe IS6110 insertion sequence, a member of the IS3 family of insertion sequences, was found to be specific to the Mycobacterium tuberculosis complex (MTBC). Although IS6110 has been extensively characterized as a transposable genetic marker, the evolutionary history of its own transposase-encoding sequence has not, to the best of our knowledge, been investigated.Methodology/Principal FindingsHere we explored the evolution of the IS6110 sequence by analysing the genetic variability and the selective forces acting on its transposase-encoding open reading frames (ORFs) A and B (orfA and orfB). For this purpose, we used a strain collection consisting of smooth tubercle bacilli (STB), an early branching lineage of the MTBC, and present-day M. tuberculosis strains representing the full breadth of genetic diversity in Tunisia. In each ORF, we found a major haplotype that dominated over a flat distribution of rare descendent haplotypes, consisting mainly of single- and double-nucleotide variant singletons. The predominant haplotypes consisted of both ancestral and present-day strains, suggesting that IS6110 acquisition predated the emergence of the MTBC. There was no evidence of recombination and both ORFs were subjected to strict purifying selection, as demonstrated by their dN/dS ratios (0.29 and 0.51, respectively), as well as their significantly negative Tajima’s D statistics. Strikingly, the purifying selection acting on orfA proved much more stringent, suggesting its critical role in regulating the transpositional process. Maximum likelihood analyses further excluded any possibility of positive selection acting on single amino acid residues.Conclusions/SignificanceTaken together our data fit with an evolutionary scenario according to which the observed variability pattern of the IS6110 transposase-encoding ORFs is generated mainly through random point mutations that accrued on a functionally optimal IS6110 copy, whose acquisition predated the emergence of the MTBC complex. Background selection acting against deleterious mutations led to an excess of low-frequency variants.

Insertion Sequence IS26 Reorganizes Plasmids in Clinically Isolated Multidrug-Resistant Bacteria by Replicative Transposition.

ABSTRACTCarbapenemase-producing Enterobacteriaceae (CPE), which are resistant to most or all known antibiotics, constitute a global threat to public health. Transposable elements are often associated with antibiotic resistance determinants, suggesting a role in the emergence of resistance. One insertion sequence, IS26, is frequently associated with resistance determinants, but its role remains unclear. We have analyzed the genomic contexts of 70 IS26 copies in several clinical and surveillance CPE isolates from the National Institutes of Health Clinical Center. We used target site duplications and their patterns as guides and found that a large fraction of plasmid reorganizations result from IS26 replicative transpositions, including replicon fusions, DNA inversions, and deletions. Replicative transposition could also be inferred for transposon Tn4401, which harbors the carbapenemase blaKPC gene. Thus, replicative transposition is important in the ongoing reorganization of plasmids carrying multidrug-resistant determinants, an observation that carries substantial clinical and epidemiological implications for understanding how such extreme drug resistance phenotypes evolve.

Different IncI1 plasmids from Escherichia coli carry ISEcp1-blaCTX-M-15 associated with different Tn2-derived elements

The blaCTX-M-15 gene, encoding the globally dominant CTX-M-15 extended-spectrum β-lactamase, has generally been found in a 2.971-kb ISEcp1-blaCTX-M-15-orf477Δ transposition unit, with ISEcp1 providing a promoter. In available IncF plasmid sequences from Escherichia coli, this transposition unit interrupts a truncated copy of transposon Tn2 that lies within larger multiresistance regions. In E. coli, blaCTX-M-15 is also commonly associated with IncI1 plasmids and here three such plasmids from E. coli clinical isolates from western Sydney 2006–2007 have been sequenced. The plasmid backbones are organised similarly to those of other IncI1 plasmids, but have insertions and/or deletions and sequence differences. Each plasmid also has a different insertion carrying blaCTX-M-15. pJIE113 (IncI1 sequence type ST31) is almost identical to plasmids isolated from the 2011 E. coli O104:H4 outbreak in Europe, where the typical blaCTX-M-15 transposition unit interrupts a complete Tn2 inserted directly in the plasmid backbone. In the novel plasmid pJIE139 (ST88), ISEcp1-blaCTX-M-15-orf477Δ lies within a Tn2/3 hybrid transposon. Homologous recombination could explain movement of ISEcp1-blaCTX-M-15-orf477Δ between copies of Tn2 on IncF and IncI1 plasmids and generation of the Tn2/3 hybrid. pJIE174 (ST37) is almost identical to pESBL-12 from the Netherlands and in these plasmids blaCTX-M-15 is flanked by two copies of IS26 that truncate the transposition unit within a larger region bounded by the ends of Tn2. blaCTX-M-15 and the associated ISEcp1-derived promoter may be able to move from this structure by the actions of IS26, independently of both ISEcp1 and Tn2.

Tn6026 and Tn6029 are found in complex resistance regions mobilised by diverse plasmids and chromosomal islands in multiple antibiotic resistant Enterobacteriaceae

Transposons flanked by direct copies of IS26 are important contributors to the evolution of multiple antibiotic resistance. Tn6029 and Tn6026 are examples of composite transposons that have become widely disseminated on small and large plasmids with different incompatibility markers in pathogenic and commensal Escherichia coli and various serovars of Salmonella enterica. Some of the plasmids that harbour these transposons also carry combinations of virulence genes. Recently, Tn6029 and Tn6026 and derivatives thereof have been found on chromosomal islands in both established and recently emerged pathogens. While Tn6029 and Tn6026 carry genes encoding resistance to older generation antibiotics, they also provide a scaffold for the introduction of genes encoding resistance to a wide variety of clinically relevant antibiotics that are mobilised by IS26. As a consequence, Tn6029 and Tn6026 or variants are likely to increasingly feature in complex resistance regions in multiple antibiotic resistant Enterobacteriaceae that threaten the health of humans and food production animals.

Genetic diversity of the Mycobacterium tuberculosis Beijing family in Brazil and Mozambique and relation with infectivity and induction of necrosis in THP-1 cells

The success of the Mycobacterium tuberculosis Beijing (MtbB) lineage in different geographical regions has been attributed to high transmission, increased virulence, drug resistance and rapid adaptation to the host. In some countries of secondary MtbB dispersion like South Africa and Peru, rising prevalence of the Beijing strains is registered. However, in neighboring countries to affected regions such as Mozambique and Brazil, respectively, the prevalence of these strains is still low and this could be due to biological particularities of the circulating MtbB strains and/or differentiated host susceptibility. To characterize genetically and phenotypically MtbB strains isolated in Brazil (n = 8) and Mozambique (n = 17). This is a descriptive study of genotypes of the MtbB isolates, determined by spoligotyping, MIRU-VNTR typing, analysis of the IS6110 copy number in the NTF region and screening for mutations in mutT2, mutT4, rpoB, katG and pks 15/1 genes. Virulence-associated properties of the studied isolates were verified in the in vitro model of infection of human THP-1 cells. The genotypes defined by the 24VNTRs were distinct for all isolates included in this study and presented an HGDI of 0.997. The VNTR patterns with seven copies of MIRU26 and seven copies of QUB26, representative for the previously described MtbB genotype B0, dominant in Russia, were detected in 38.5% of the studied isolates. In addition, all isolates presented RD105 deletion and a 7 bp insertion in pks15/1 gene. Almost all tested strains belonged to the RD181 sublineage, with the exception of two strains from Mozambique of RD150 sublineage. Combined analysis of the NTF region integrity and mutations in mutT genes showed that 62.5% and 47% of isolates obtained in Brazil and Mozambique, respectively, were of the ancestral genotype. The virulence index of the ancient isolates, evaluated in the THP-1 cells, was significantly lower than that of the modern genotype group. These data demonstrate genotype particularities of the Beijing strains isolated in Brazil and Mozambique, two countries of low prevalence of the MtbB lineage in local Mtb populations. In contrast to the neighboring countries with high prevalence of the MtbB strains of modern sublineage, significant proportions of the isolates obtained in Brazil and Mozambique were presented by the strains of the ancient sublineage. Our data suggest that lower virulence of the ancient strains, compared with the modern strains, could be involved in the slow spread of the MtbB strains in some regions.

Movement of IS26-associated antibiotic resistance genes occurs via a translocatable unit that includes a single IS26 and preferentially inserts adjacent to another IS26.

ABSTRACTThe insertion sequence IS26 plays a key role in disseminating antibiotic resistance genes in Gram-negative bacteria, forming regions containing more than one antibiotic resistance gene that are flanked by and interspersed with copies of IS26. A model presented for a second mode of IS26 movement that explains the structure of these regions involves a translocatable unit consisting of a unique DNA segment carrying an antibiotic resistance (or other) gene and a single IS copy. Structures resembling class I transposons are generated via RecA-independent incorporation of a translocatable unit next to a second IS26 such that the ISs are in direct orientation. Repeating this process would lead to arrays of resistance genes with directly oriented copies of IS26 at each end and between each unique segment. This model requires that IS26 recognizes another IS26 as a target, and in transposition experiments, the frequency of cointegrate formation was 60-fold higher when the target plasmid contained IS26. This reaction was conservative, with no additional IS26 or target site duplication generated, and orientation specific as the IS26s in the cointegrates were always in the same orientation. Consequently, the cointegrates were identical to those formed via the known mode of IS26 movement when a target IS26 was not present. Intact transposase genes in both IS26s were required for high-frequency cointegrate formation as inactivation of either one reduced the frequency 30-fold. However, the IS26 target specificity was retained. Conversion of each residue in the DDE motif of the Tnp26 transposase also reduced the cointegration frequency.

Dissemination of a clone carrying a fosA3-harbouring plasmid mediates high fosfomycin resistance rate of KPC-producing Klebsiella pneumoniae in China

Fosfomycin has been proposed as an adjunct to other active agents for treating KPC-producing Klebsiella pneumoniae infections. This study aimed to investigate the prevalence of fosfomycin resistance and plasmid-mediated resistance determinants among KPC-producing K. pneumoniae isolates from clinical samples in China. In total, 278 KPC-producing and 80 extended-spectrum β-lactamase (ESBL)-producing (non-KPC-producing) clinical K. pneumoniae isolates were collected in 12 hospitals from 2010 to 2013. Fosfomycin susceptibility testing was carried out using the agar dilution method. Phylogenetic clonal patterns were revealed by pulsed-field gel electrophoresis (PFGE). Isolates were screened for plasmid-mediated fosfomycin resistance genes (fosA, fosA3 and fosC2) by PCR amplification. A plasmid was completely sequenced by next-generation sequencing. The fosfomycin resistance rate in KPC-producers (60.8%; 169/278) was significantly higher than in ESBL-producers (12.5%; 10/80). In addition, 94 KPC-producing isolates were positive for fosA3 and most of them were clonally related. A 23939-bp plasmid (pFOS18) co-harbouring fosA3 and blaKPC-2 was completely sequenced, revealing that the fosA3 gene was flanked by two copies of IS26; however, blaKPC-2 was located on a Tn3–Tn4401 integration structure. Although the fosA3 and blaKPC-2 genes are located on different transposon systems, they are able to spread together worldwide through plasmid transfer. Dissemination of the clone carrying the fosA3-harbouring plasmid mediates the high fosfomycin resistance rate of KPC-producing K. pneumoniae in China. Fosfomycin as an alternative option for treating infections caused by KPC-producing K. pneumoniae should not be recommended in hospitals in which fosfomycin-resistant clonal dissemination is emerging.