Replicon typing of plasmids encoding resistance to newer [beta]-lactams.Polymerase chain reaction-based replicon rep·li·con n. A genetic element that undergoes replication as an autonomous unit. typing represents a novel method to describe the dissemination and follow the evolution of resistance plasmids. We used this approach to study 26 epidemiologically unrelated Enterobacteriaceae and demonstrate the dominance of incompatibility (Inc) A/C or Inc N-related plasmids carrying some emerging resistance determinants to extended-spectrum cephalosporins Cephalosporins Definition Cephalosporins are medicines that kill bacteria or prevent their growth. Purpose Cephalosporins are used to treat infections in different parts of the body—the ears, nose, throat, lungs, sinuses, and and carbapenems. ********** Understanding the molecular epidemiology molecular epidemiology Molecular medicine An evolving field that combines the tools of standard epidemiology–case studies, questionnaires and monitoring of exposure to external factors with the tools of molecular biology–eg, restriction endonucleases, of resistance plasmids has been a major issue since scientists became aware of plasmids' role in the spread of antimicrobial drug resistance. However, understanding this epidemiology has been complex because of the diversity and promiscuity Promiscuity See also Profligacy. Anatol constantly flits from one girl to another. [Aust. Drama: Schnitzler Anatol in Benét, 33] Aphrodite promiscuous goddess of sensual love. [Gk. Myth. of these elements. The plasmid replication system, which dictates the plasmid's behavior (host range, copy number) is the major plasmid landmark from a biologic standpoint; it is used for plasmid classification and identification (1). Plasmids were originally classified in incompatibility (Inc) groups (2). Inc is a manifestation of plasmid relatedness based on commonality of replication controls. The standard procedure for determining Inc groups requires laborious hands-on work, multiple conjugation (Biol.) a coalescence of many cells (as where an indefinite number of amœboid cells flow together into a single mass) from which conjugation proper and even fertilization may have been evolved. See also: Multiple , transformation assays, or hybridization hybridization /hy·brid·iza·tion/ (hi?brid-i-za´shun) 1. crossbreeding; the act or process of producing hybrids. 2. molecular hybridization 3. experiments (1-3). Our objective of understanding the relationship among resistance plasmids prompted us to develop a polymerase chain reaction polymerase chain reaction (pŏl`ĭmərās') (PCR), laboratory process in which a particular DNA segment from a mixture of DNA chains is rapidly replicated, producing a large, readily analyzed sample of a piece of DNA; the process is (PCR PCR polymerase chain reaction. PCR abbr. polymerase chain reaction Polymerase chain reaction (PCR) )-based replicon typing method (4). Our study has 2 aims: 1) to investigate phylogenetic phy·lo·ge·net·ic adj. 1. Of or relating to phylogeny or phylogenetics. 2. Relating to or based on evolutionary development or history. relatedness among plasmids carrying extended-spectrum cephalosporin cephalosporin (sĕf'əlōspôr`ĭn), any of a group of more than 20 antibiotics derived from species of fungi of the genus Cephalosporium and closely related chemically to penicillin. Cephalosporins, e.g. (ESC See escape character and escape key. See also ESC/P. ESC - escape ) and carbapenem resistance determinants emerging in 3 different countries (Greece, Italy, and the United States United States, officially United States of America, republic (2005 est. pop. 295,734,000), 3,539,227 sq mi (9,166,598 sq km), North America. The United States is the world's third largest country in population and the fourth largest country in area. ) and 2) to ascertain the sensitivity of the method. The Study PCR-based replicon typing was applied to type the resistance plasmids carried by 26 Escherichia coli Escherichia coli (ĕsh'ərĭk`ēə kō`lī), common bacterium that normally inhabits the intestinal tracts of humans and animals, but can cause infection in other parts of the body, especially the urinary tract. transconjugants or transformants obtained from epidemiologically unrelated clinical isolates of Enterobacteriaceae associated with community- or hospital-acquired infections Hospital-Acquired Infections Definition A hospital-acquired infection is usually one that first appears three days after a patient is admitted to a hospital or other health care facility. in the United States or southern Europe Southern Europe or sometimes Mediterranean Europe is a region of the European continent. There is no clear definition of the term which can vary depending on whether geographic, cultural, linguistic or historical factors are taken into account. (Italy and Greece). The resistance plasmids carried genes encoding [beta]-lactamases of Ambler class A (SHV-12), B (VIM-1 or VIM-4), and C (CMY-2, CMY-4, or CMY-13) (Table), which represent key emerging resistance determinants to ESC and carbapenems. Eighteen primer pairs were used to perform 5 multiplex and 3 simplex PCRs, which recognized FIA FIA feline infectious anemia. , FIB fib n. An insignificant or childish lie. intr.v. fibbed, fib·bing, fibs To tell a fib. See Synonyms at lie2. , FIC FIC First International Computer FIC Fogarty International Center (John E. Fogarty International Center for Advanced Study in the Health Sciences; National Institutes of Health) FIC Fellowship for Intentional Community , HI1, HI2, I1-I[gamma], L/M L/M low and moderate (income levels) , N, P, W, T, A/C, K, B/O B/O Battery Operated B/O Breakout B/O Buyout (auctions) B/O Back Order B/O Biological Opinion B/O Blocking Oscillator B/O Broken Object B/O Budget Outlay B/O Budget Obligation , X, Y, and FII FII Federated Investors Inc. (Pittsburgh, PA) FII Foreign Institutional Investor FII Falling Into Infinity (Dream Theater album) FII Fundación Instituto de Ingeniería replicons (4). All amplified replicons were sequenced by standard procedures and used as specific probes to confirm the replicon typing results by Southern blot hybridization Southern blot hybridization Southern blotting Molecular biology A method delineated by EM Southern for detecting and manipulating specific DNA sequences previously separated by gel electrophoresis. on purified plasmid DNA (data not shown). The plasmid donors from the United States consisted of 4 previously characterized ESC-resistant Salmonella isolates submitted to the National Antimicrobial Resistance Monitoring System (NARMS NARMS National Antimicrobial Resistance Monitoring System NARMS National Association of Rug Makers and Sculptors ) from 1996 to 1998 (12) and 6 ESC-resistant Escherichia coli O157:H7 isolates collected by NARMS from 2000 to 2001 (5). During the study periods, participating state and local public health laboratories forwarded every tenth non-Typhi type Salmonella and every fifth E. coli E. coli: see Escherichia coli. E. coli in full Escherichia coli Species of bacterium that inhabits the stomach and intestines. E. coli can be transmitted by water, milk, food, or flies and other insects. O157 isolate they received to the Centers for Disease Control and Prevention Centers for Disease Control and Prevention (CDC), agency of the U.S. Public Health Service since 1973, with headquarters in Atlanta; it was established in 1946 as the Communicable Disease Center. for susceptibility testing. This collection includes representatives from sporadic and outbreak infections (5,12). The 6 Salmonella and 4 E. coli plasmid donors selected for this study were a small sample of epidemiologically unrelated isolates representative of those carrying a [bla.sub.CMT-2] [beta]-lactamase gene on plasmids classified as type A or B on the basis of the [bla.sub.CMY-2] hybridization pattern (6,13). The PCR-based replicon typing method assigned the A/C and I1 replicons to type A and type B plasmids, respectively (Table), which was confirmed by DNA sequencing. The I1-type amplicon sequences were identical to the R64 IncI1 reference plasmid (no. AP005147), whereas the A/C-type amplicon sequences exhibited 26 nucleotide (nt) substitutions with respect to the RA1 IncA/C reference plasmid (no. X73674), which caused 3 amino acid amino acid (əmē`nō), any one of a class of simple organic compounds containing carbon, hydrogen, oxygen, nitrogen, and in certain cases sulfur. These compounds are the building blocks of proteins. variations. Therefore, the A/C-replicon from the US plasmids may represent a new replicon variant, which we designated repA/[C.sub.2] (DNA sequence DNA sequence Genetics The precise order of bases–A,T,G,C–in a segment of DNA, gene, chromosome, or an entire genome. See Base pair, Base sequence analysis, Chromosome, Gene, Genome. released under EMBL EMBL European Molecular Biology Laboratory EMBL Eniwetok Marine Biological Laboratory accession no. AM087198). The Figure shows conserved PstI restriction profiles obtained for the A/[C.sub.2] plasmids that are different from those exhibited by the I1 plasmids. [FIGURE OMITTED] The plasmid donors from Italy consisted of 7 multidrug-resistant isolates of various species of Enterobacteriaceae carrying either [bla.sub.SHV-12] or [bla.sub.CMY-4] and [bla.sub.VIM-4] plasmidbome [beta]-lactamase genes (Table). These isolates had been collected from 2002 to 2003 at 4 different hospitals in northern or central Italy (7,8) and were epidemiologically unrelated, except for IT-VA416/02 and IT-VA417/02, which were from the same patient (7). PCR replicon typing of the 5 [bla.SHV-12]--carrying plasmids detected 3 repFII (100% identical to the reference sequence no. M33752), 1 repI1 (100% identical to the R64 plasmid), and 1 repA/[C.sub.1] (99% homologous homologous /ho·mol·o·gous/ (ho-mol´ah-gus) 1. corresponding in structure, position, origin, etc. 2. allogeneic. ho·mol·o·gous adj. 1. to the RA1 plasmid) (Table), suggesting mobilization of this gene among different plasmid scaffolds. The [bla.sub.SHV-12] plasmids showed different PstI restriction patterns, which confirmed their diversity (Figure). The 2 plasmids carrying [bla.sub.VIM-4] and [bla.sub.CMY-4] were assigned by PCR replicon typing to the A/C type. The sequence of these replicons showed the same 26 characteristic nucleotide substitutions of the A/[C.sub.2]-replicon identified in the US plasmids. These 2 A/[C.sub.2]-plasmids showed an apparently identical PstI restriction profile (data not shown), which was also very similar to that of some USA [bla.sub.CMY (Cyan Magenta Yellow) The color space used for printing. In theory, equal amounts of all three colors produce black. In practice, a separate black ink is required for quality printing. See CMYK. .2] plasmids (see the 2039 and 3977 US plasmids and the Italian VA416/02 plasmid in the Figure). The 2 Italian A/[C.sub.2] plasmids, in addition to [bla.sub.CMY-4]. (which is a [bla.sub.CMY-2] variant different by only a single nucleotide substitution), also carried the [bla.sub.VIM-4] carbapenemase gene, which has not been reported on [bla.sub.CMY-2]--carrying plasmids from the United States and may represent a novel acquisition. These findings indicate intercontinental spread of these plasmids and novel acquisition of resistance genes. The plasmid donors from Greece consisted of a collection of 7 Klebsiella pneumoniae Klebsiella pneu·mo·ni·ae n. Friedlander's bacillus. isolates carrying the [bla.sub.VIM-1] gene (9) and 2 E. coli isolates carrying [bla.sub.VIM-1] and [bla.sub.CMY-13] genes (10). These isolates, randomly collected from 5 different hospitals in Athens and Piraeus from 2001 to 2003, are representative of the VIM-1-producing isolates circulating in Greece. No repetitive samples were taken from patients. All isolates exhibited decreased susceptibility to carbapenems. Restriction analysis of these plasmids classified them into 6 different groups on the basis of their restriction profiles (Figure). By replicon typing, all of these plasmids were assigned to the same repN-type replicon, which exhibited 2-nt point mutations (99% homology homology (hōmŏl`əjē), in biology, the correspondence between structures of different species that is attributable to their evolutionary descent from a common ancestor. ) in respect to the R46 IncN reference plasmid (no. NC_003292), an indication that they were phylogenetically phy·lo·ge·net·ic adj. 1. Of or relating to phylogeny or phylogenetics. 2. Relating to or based on evolutionary development or history: a phylogenetic classification of species. related and probably evolved from a common ancestor. Although one might expect similar plasmid scaffolds to exist among isolates in Greece and Italy because of geographic proximity, this was not the case. This finding explains the great variability of resistance plasmids carrying different combinations of resistance genes. Since the origin of replication The origin of replication (also called the replication origin) is a particular DNA sequence at which DNA replication is initiated. DNA replication may proceed from this point bidirectionally or unidirectionally. is a constant and conserved part of a plasmid, replicon typing focused on this portion of the plasmid is a more sensitive and specific method for identifying phylogenetically related plasmids than restriction-based analysis of the entire plasmid. This fact is probably due to the presence of multiple mobile elements (IS elements, transposons Transposons Types of transposable elements which comprise large discrete segments of deoxyribonucleic acid (DNA) capable of moving from one chromosome site to a new location. , integrons) that can mediate rearrangements of the plasmid scaffolds, which leads to the formation of apparently divergent plasmids. In fact, this phenomenon was demonstrated for the GR-541 plasmid that contains multiple copies of insertion sequences and other mobile genetic elements Mobile genetic elements (MGE) are a type of DNA that can move around within the genome. They include:
Conclusions A PCR-based replicon typing approach was successfully applied to relevant resistance plasmids. Coupled with sequencing, the approach allowed high-resolution typing of the plasmid replicons. Typing results provided original insights into the molecular epidemiology of resistance plasmids. For instance, the [bla.sub.CMY-2]--carrying plasmid circulating in the United States was also detected in Europe in the form of a derivative that also carries the VIM-4 car-bapenemase determinant. This finding demonstrates that plasmids carrying resistance to clinically relevant antimicrobial agents can spread worldwide among bacteria responsible for both nosocomial nosocomial /noso·co·mi·al/ (nos?o-ko´me-il) pertaining to or originating in a hospital. nos·o·co·mi·al adj. 1. Of or relating to a hospital. 2. and community-acquired infections. The heterogeneity among Italian plasmids encoding SHV-12 (the most prevalent SHV-type extended-spectrum [beta]-lactamase in this country) (15) suggests a notable potential for this determinant to spread among different plasmid replicons. On the other hand, replicon typing indicated that the VIM-1-encoding plasmids from Greece were all related despite their different restriction profiles, which points out the common origin of these plasmids. The [bla.sub.CMY-13] gene from Greece is located on the repN plasmid, whereas Italy and the United States share the A/[C.sub.2] plasmid as a vehicle of the [bla.sub.CMY] gene, despite their geographic distance. Further research is necessary to determine the influences on plasmid trafficking as well as further similarities and differences. Replicon identification may provide useful clues to the evolution of these resistant plasmids. The ability to trace and screen plasmids by PCR may facilitate further understanding of the horizontal transfer of antimicrobial drug resistance. This work is supported by the MED-NET-VET (FOOD-CT-2004-506122, WP09) and DREPS2 (LSHM-CT-2005-018705) contracts with the European Commission and European Research Network within the Training and Mobility of Researchers program (HPRN-CT-2002-00264). References (1.) Novick RP. Plasmid incompatibility. Microbiol Rev. 1987;51:381-95. (2.) Datta N, Hughes VM. Plasmids of the same Inc groups in Enterobacteria en·ter·o·bac·te·ri·um n. pl. en·ter·o·bac·te·ri·a Any of various gram-negative rod-shaped bacteria of the family Enterobacteriaceae that includes some pathogens of plants and animals, such as the colon bacillus and salmonella. before and after the medical use of antibiotics. Nature. 1983;306:616-7. (3.) Couturier M, Bex F, Bergquist PL, Maas WK. Identification and classification of bacterial plasmids. Microbiol Rev. 1988;52:375-95. (4.) Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods. 2005;63:219-28. (5.) Whichard JM, Joyce K, Fey P, Nelson JM, Angulo F J, Barrett TJ. [beta]lactam resistance and Enterobacteriaceae, United States. Emerg Infect Dis. 2005;11:1464-6. (6.) Carattoli A, Tosini F, Giles WP, Rupp ME, Hinrichs SH, Angulo FJ, et al. Characterization of plasmids carrying CMY-2 from expanded-spectrum cephalosporin-resistant Salmonella isolated in the United States between 1996 and 1998. Antimicrob Agents Chemother. 2002;46:1269-72. (7.) Luzzaro F, Docquier JD, Colinon C, Endimiani A, Lombardi G, Amicosante G, et al. Emergence in Klebsiella pneumoniae and Enterobacter cloacae clinical isolates of the VIM-4 metallo-[beta]-lactamase encoded by a conjugative plasmid con·ju·ga·tive plasmid n. A plasmid that can move from one cell to another during the process of conjugation. . Antimicrob Agents Chemother. 2004;48:648 50. (8.) Luzzaro F, Mezzatesta M, Mugnaioli C, Perilli M, Stefani S, Amicosante G, et ah Trends in the production of extended-spectrum [beta]-lactamases among enterobacteria of medical interest. Report of the second Italian nationwide survey. J Clin Microbiol. 2006; 44: 1659-64. (9.) Giakkoupi P, Xanthaki A, Kanelopoulou M, Vlahaki A, Miriagou V, Kontou S, et ah VIM-1 metallo-[beta]-lactamase-producing Klebsiella pneumoniae strains in Greek Hospitals. J Clin Microbioh 2003;41:3893-6. (10.) Miriagou V, Tzelepi E, Gianneli D, Tzouvelekis LS. Escherichia coli with a self-transferable, multi-resistant plasmid coding for the metallo-[beta]-lactamase VIM-1. Antimicrob Agents Chemother. 2003; 47:395-7. (11.) Clinical and Laboratory Standards Institute. Performance standards for antimicrobial disk susceptibility tests; approved standard. 9th ed. Wayne (PA): The Institute; 2006. (12.) Dunne EF, Fey PD, Kludt P, Reporter R, Mostashari F, Shillam P, et al. Emergence of domestically acquired ceftriaxone-resistant Salmonella infections associated with AmpC beta-lactamase. JAMA JAMA abbr. Journal of the American Medical Association . 2000;284:3151-6. (13.) Giles WE Benson AK, Olson ME, Hutkins RW, Whichard JM, Winokur PL, et ah DNA sequence analysis of regions surrounding blaCMY-2 from multiple Salmonella plasmid backbones. Antimierob Agents Chemother. 2004;48:2845-52. (14.) Miriagou V, Carattoli A, Tzelepi E, Villa L, Tzouvelekis LS. IS26-associated In4-type integrons forming multiresistance loci loci [L.] plural of locus. loci Plural of locus, see there in enterobacterial plasmids. Antimicrob Agents Chemother. 2005;49:3541-3. (15.) Perilli M, Dell'Amico E, Segatore B, De Massis MR, Bianchi C, Luzzaro F, et al. Molecular characterization of extended-spectrum [beta]-lactamases produced by nosocomial isolates of Enterobacteriaceae from an Italian nationwide survey. J Clin Microbiol. 2002;40:611-4. Alessandra Carattoli, * Vivi Miriagou, ([dagger]) Alessia Bertini, * Alexandra Loli, ([dagger]) Celine Colinon, ([double dagger]) Laura Villa, * Jean M. Whichard, ([section]) and Gian Maria Rossolini ([double dagger]) * Istituto Superiore di Sanita, Rome, Italy; 1Institute Pasteur, Athens, Greece; ([double dagger]) Universita di Siena, Siena, Italy; and ([section]) Centers for Disease Control and Prevention, Atlanta, Georgia, USA Address for correspondence: Alessandra Carattoli, Department of Infectious, Parasitic, Immune-mediated Diseases, Istituto Superiore di Sanita, Rome, Viale Regina Elena 299, 00161 Rome, Italy; email: alecara@iss.it Dr Carattoli is a researcher in the Department of Infectious, Parasitic, Immune-mediated Diseases at the Istituto Superiore di Sanita. Her areas of research interest include the genetic basis of multidrug resistance multidrug resistance, n the adaptation of tumor cells or infectious agents to resist chemotherapeutic agents. and plasmid characterization of gram-negative bacteria.
Table. Phenotypic and genetic characteristics of plasmids and
transformant/transconjugant strains analyzed in this study
Transferred resistance
traits in transconjugants
Original strain Species and serovar or transformants *
USA-4204 Salmonella enterica AmpCazCroCtxFox
serovar Typhimurium
USA-2039 S. Typhimurium AmpCazCroCtxFoxGmTo
USA-3977 S. Typhimurium AmpCazCroCtxFox
USA-8401 Escherichia coli 0157:H7 AmpCazCroCtxFox
USA-8749 E. coli O157:H7 AmpCazCroCtxFox
USA-8868 E. coli O157:H7 AmpCazCroCtxFox
USA-1091 E. coli O157:H7 AmpCazCroCtxFox
USA-7546 E. coli O157:H7 AmpCazCroCtxFox
USA-11371 E. coli O157:H7 AmpAtmCazCroCtxFox
USA-1358 S. Thompson AmpAtmCazCroCtxFox
IT-VA416/02 Klebsiella pneumoniae AmpAtmCazCroCtxFoxlpmGmTo
IT-VA417/02 Enterobacter cloacae AmpAtmCazCroCtxFoxlpmGmTo
IT-FI045T Enterobacter aerogenes AmpAtmCaz
IT-FI008T E. coli AmpAtmCazTo
IT-BG003T Serratia marcescens AmpAtmCazTo
IT-NO003T Klebsiella oxytoca AmpAtmCaz
IT-BG017T K. pneumoniae AmpAtmCazCroCtx
GR-541 E. coli AmpAtmCazCtxCroFoxlpmTo
GR-116 E. coli AmpAtmCazCroCtxFoxlpmGmTo
GR-700 K. pneumoniae AmpCazCroCtxFoxlpmGmTo
GR-2564 K. pneumoniae AmpCazCroCtxFoxlpmTo
GR-1943 K. pneumoniae AmpCazCroCtxFoxlpmTo
GR-1955 K. pneumoniae AmpCazCroCtxFoxlpmTo
GR-5866 K. pneumoniae AmpCazCroCtxFoxlpmTo
GR-51395 K. pneumoniae AmpCazCroCtxFoxlpmTo
GR-6/100 K. pneumoniae AmpCazCroCtxFoxlpmTo
bla genes identified on Replicons detected
Original strain transferred plasmids by PCR ([dagger])
USA-4204 CMY-2-type A A/[C.sub.2]
USA-2039 CMY-2-type A A/[C.sub.2]
USA-3977 CMY-2-type A A/[C.sub.2]
USA-8401 CMY-2-type A A/[C.sub.2]
USA-8749 CMY-2-type A A/[C.sub.2]
USA-8868 CMY-2-type A A/[C.sub.2]
USA-1091 CMY-2-type A A/[C.sub.2]
USA-7546 CMY-2-type A A/[C.sub.2]
USA-11371 CMY-2-type B I1
USA-1358 CMY-2-type B I1
IT-VA416/02 VIM-4, CMY-4 A/[C.sub.2]
IT-VA417/02 VIM-4, CMY-4 A/[C.sub.2]
IT-FI045T SHV-12 FII
IT-FI008T SHV-12 FII
IT-BG003T SHV-12 FII
IT-NO003T SHV-12 A/[C.sub.1]
IT-BG017T SHV-12 I1
GR-541 VIM-1, CMY13 N
GR-116 VIM-1, CMY13 N
GR-700 VIM-1 N
GR-2564 VIM-1 N
GR-1943 VIM-1 N
GR-1955 VIM-1 N
GR-5866 VIM-1 N
GR-51395 VIM-1 N
GR-6/100 VIM-1 N
* Transconjugants or transformants were obtained by conjugation on
solid medium or electroporation, respectively, with E. coli (strains
DH5-[alpha] or DH10B or 26R793 as recipients), as described previously
(5-10). Antimicrobial drug susceptibility of the transconjugants or
transformants was determined by disk diffusion as recommended by the
Committee on Clinical and Laboratory Standards (11) with the following
drugs: Amp, ampicillin; Atm, aztreonam; Caz, ceftazidime; Cro,
ceftriaxone; Ctx, cefotaxime, Fox, cefoxitin; Ipm, imipenem; Gm,
gentamicin; To, tobramycin. Transferred resistance traits were inferred
by a detectable reduction of susceptibility to the drug in the
transconjugant or transformant, compared to the E. coli recipient.
([dagger]) Discrimination between A/[C.sub.1] and A/[C.sub.2] replicons
was performed by DNA sequencing of the amplicon obtained by replicon
typing.
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