Nosocomial infections caused by antibiotic-resistant Klebsiella pneumoniae are emerging as a major health problem worldwide, while community-acquired K. pneumoniae infections present with a range of diverse clinical pictures in different geographic areas. In particular, an invasive form of K. pneumoniae that causes liver abscesses was first observed in Asia and then was found worldwide. We are interested in how differences in gene content of the same species result in different diseases. Thus, we sequenced the whole genome of K. pneumoniae NTUH-K2044, which was isolated from a patient with liver abscess and meningitis, and analyzed differences compared to strain MGH 78578, which was isolated from a patient with pneumonia. Six major types of differences were found in gene clusters that included an integrative and conjugative element, clusters involved in citrate fermentation, lipopolysaccharide synthesis, and capsular polysaccharide synthesis, phage-related insertions, and a cluster containing fimbria-related genes. We also conducted comparative genomic hybridization with 15 K. pneumoniae isolates obtained from community-acquired or nosocomial infections using tiling probes for the NTUH-K2044 genome. Hierarchical clustering revealed three major groups of genomic insertion-deletion patterns that correlate with the strains' clinical features, antimicrobial susceptibilities, and virulence phenotypes with mice. Here we report the whole-genome sequence of K. pneumoniae NTUH-K2044 and describe evidence showing significant genomic diversity and sequence acquisition among K. pneumoniae pathogenic strains. Our findings support the hypothesis that these factors are responsible for the changes that have occurred in the disease profile over time.
A plasmid containing the qnrS quinolone resistance determinant and the gene encoding the SHV-2 -lactamase has been discovered from a clinical Klebsiella pneumoniae strain isolated in Taiwan. The complete 98-kb sequence of this plasmid, designated pK245, was determined by using a whole-genome shotgun approach. Transfer of pK245 conferred low-level resistance to fluoroquinolones in electroporant Escherichia coli epi300. The sequence of the immediate region surrounding qnrS in pK245 is nearly identical (>99% identity) to those of pAH0376 from Shigella flexneri and pINF5 from Salmonella enterica serovar Infantis, the two other qnrS-carrying plasmids reported to date, indicating a potential common origin. Other genes conferring resistance to aminoglycosides (aacC2, strA, and strB), chloramphenicol (catA2), sulfonamides (sul2), tetracycline (tetD), and trimethoprim (dfrA14) were also detected in pK245. The dfrA14 gene is carried on a class I integron. Several features of this plasmid, including three separate regions containing putative replicons, a partitioning-control system, and a type II restriction modification system, suggest that it may be able to replicate and adapt in a variety of hosts. Although no critical conjugative genes were detected, multiple insertion sequence elements were found scattered throughout pK245, and these may facilitate the dissemination of the antimicrobial resistance determinants. We conclude that pK245 is a chimera which acquired its multiple antimicrobial resistance determinants horizontally from different sources. The identification of pK245 plasmid expands the repertoire of the coexistence of quinolone and extended-spectrum--lactam resistance determinants in plasmids carried by various species of the family Enterobacteriaceae in different countries.Plasmid-mediated quinolone resistance was first identified in a clinical Klebsiella pneumoniae strain in the United States in 1998 (29). The gene responsible for this mechanism, qnrA, was later characterized (43). Several QnrA-and QnrB-like proteins have since been identified in different species of the family Enterobacteriaceae, including Escherichia coli, Enterobacter cloacae, and K. pneumoniae from the United States, Europe, and Asia (6,21,22,27,32,36,37,44,45). Another qnr gene, qnrS, was recently found on a transferable plasmid from a clinical Shigella flexneri isolate in Japan (19) and has subsequently been found in a Salmonella enterica serovar Infantis isolate of avian origin (23). Although many qnrA-and qnrB-positive plasmids were also found to carry genes encoding extended-spectrum -lactamases (ESBLs) (21, 32, 34), no ESBLs were found on the plasmids that carried qnrS.We recently identified a multidrug resistance plasmid from K. pneumoniae strain NK245. NK245 was isolated in January 2002 from a patient with hospital-acquired urinary tract infection. The plasmid, designated pK245, conferred resistance to several classes of antimicrobials in the E. coli electroporant, including low-level resistance to fluoroquinolones (FQs). The...
The DNA sequences of two IncHI2 plasmids, pEC-IMP and pEC-IMPQ, from metallo--lactamaseproducing Enterobacter cloacae clinical isolates were determined. The two conjugative plasmids are almost identical, but pEC-IMPQ carries an additional segment containing an orf513 (ISCR1), a truncated 3 conserved sequence, and a qnrB2. Comparative analyses provide support for the proposed ISCR1-mediated gene mobilization.Encoding a putative product of 513 amino acids, orf513 was initially identified adjacent to integrons In6 and In7 (11). Together with noncassette resistance genes, it was commonly found between truncated and full-length 3Ј conserved sequences (3Ј-CS) of class 1 integrons (9, 11). Their function remained mysterious until comparative analyses linked these so-called common region (CR) elements to a group of IS91-like insertion sequences (ISs) (13). The IS91-like ISs are a family of unusual IS elements that differ from most other IS elements in both structure and mode of transposition. They can perform rolling-circle (RC) transposition, in which a single IS element can mobilize the sequences to which it is attached (4, 12). It was proposed that orf513, later termed insertion sequence common region 1 (ISCR1), may have mobilized the nearby sequence and a truncated 3Ј-CS from one integron to the 3Ј-CS of another integron through RC transposition, thus facilitating the formation of complex class 1 integrons associated with ISCR1 (13). In addition to this putative recombinase function, the ISCR1 element has also been shown to play a role in the expression of nearby genes by providing a promoter (8, 10).ISCR1 were found to be associated with many antimicrobial resistance genes, including the plasmid-mediated quinolone resistance determinant qnr (5) as well as genes encoding resistance to chloramphenicol, trimethoprim, aminoglycosides, and -lactams (8,13,14). However, lacking the 59 base elements required for site-specific recombination, these orf513-linked genes could not have been acquired as gene cassettes. It was hypothesized that these antimicrobial resistance genes were added to the 3Ј-CS of the class 1 integron through comobilization with the nearby ISCR1 from other integrons using RC transposition and homologous recombination (1, 13).In a recent study on the prevalence of QnrA, QnrB, and QnrS among clinical isolates of Enterobacter cloacae, the association of Qnr with the IMP-8 metallo--lactamase (MBL) was investigated (15). From 56 IMP-8 MBL producers, eight qnrB-positive, bla IMP-8 -positive transconjugants and four qnrB-negative bla IMP-8 -positive transconjugants were obtained. Restriction pattern analysis on these plasmids gave very similar patterns, suggesting the occurrence of horizontal mobility of qnrB2 (15). To investigate the possible horizontal transfer mechanisms responsible for qnrB2, we have conducted complete DNA sequencing and comparative analysis on two of the plasmids, the qnrB2-positive plasmid pEC-IMPQ and the qnrB2-negative plasmid pEC-IMP.The DNA sequences of the two plasmids were deter...
fWe report the complete genome sequence of Klebsiella pneumoniae 1084, a hypermucoviscosity-negative K1 clinical strain. Sequencing and annotation revealed a 5,386,705-bp circular chromosome (57.4% G؉C content), which contains 4,962 proteincoding genes, 80 tRNA genes, and 25 rRNA genes.
A 269-kilobase conjugative plasmid, pK29, from a Klebsiella pneumoniae strain was sequenced. The plasmid harbors multiple antimicrobial resistance genes, including those encoding CMY-8 AmpC-type and CTX-M-3 extended-spectrum -lactamases in the common backbone of IncHI2 plasmids. Mechanisms for dissemination of the resistance genes are highlighted in comparative genomic analyses.Plasmid-mediated -lactamases play a key role in the increasing multidrug resistance in the Enterobacteriaceae worldwide, among which CTX-M-type extended-spectrum -lactamases (ESBLs) and AmpC-type -lactamases are two major contributors in recent years (3,14,17,19). In general, ESBLs confer resistance to oxyimino-cephalosporins but not cephamycins and are inhibited by -lactamase inhibitors, while AmpC-type -lactamases provide resistance to cephamycins and oxyimino-cephalosporins and are refractory to -lactamase inhibitors. Although reports of multiple -lactamases in a single pathogen are increasing for the Enterobacteriaceae, especially Klebsiella pneumoniae (1,9,15,22,26), the complete sequence information for a plasmid that encodes both an ESBL and an AmpC-type -lactamase has not been reported.In Taiwan, CTX-M-and SHV-type ESBLs and CMY-and DHA-type AmpC-type enzymes are the most common -lactamases that can confer resistance to extended-spectrum cephalosporins in clinical K. pneumoniae isolates (24, 26). Here we report the sequencing, annotation, and comparative genomic analysis of an IncHI2 plasmid isolated from a nosocomial K. pneumoniae strain. The plasmid carries both CTX-M-and CMY-type -lactamase genes.Three K. pneumoniae isolates from three patients were collected from the National Cheng Kung University Hospital in Taiwan during a 1-month period in 2001. These three isolates shared identical antimicrobial susceptibility and plasmid profiles (data not shown). Conjugal transfer was performed by using one of the K. pneumoniae isolates, NK29, as the donor and Escherichia coli J53 Azi r as the recipient, following a previously described protocol (11). Transconjugants were obtained at an efficiency of 10 Ϫ4 to 10 Ϫ5 transconjugants/donor at 25°C. DNA sequencing of the plasmid was determined as part of the process of sequencing the entire genome of K. pneumoniae NK29, using a shotgun approach. Sequence assembly, annotation, and analysis followed previously described protocols (4).The plasmid pK29 was determined to be a 269,674-bp circular plasmid with a GϩC content of 46%. A total of 310 open reading frames (ORFs) were identified. Since pK29 carries the backbone of the IncHI2 plasmids, we compared its nucleotide sequence with those of pR478 and pAPEC-O1-R, the IncHI2 plasmids with complete nucleotide sequences available to date. Plasmid pR478 (275 kb) was from Serratia marcescens clinical isolates, and plasmid pAPEC-O1-R (241 kb) was from avian pathogenic E. coli strains (6, 12).The common regions of the three plasmids contain essential genes for plasmid replication, maintenance, and transmission. These include the repHIA and re...
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