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armA and aminoglycoside resistance in Escherichia coli.



We report armA in an Escherichia coli pig isolate from Spain. The resistance gene was borne by self-transferable IncN plasmid pMUR050. Molecular analysis of the plasmid and of the armA locus confirmed the spread of this resistance determinant.

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A minoglycosides are used to treat a broad range of life-threatening infections in humans and animals (1,2). Thus, surveillance of resistance to these antimicrobial agents and study of the corresponding mechanisms in pathogenic bacteria remain ongoing challenges in microbial research (3). Since 1996, the Spanish Veterinary Antimicrobial Resistance Surveillance Network has monitored antimicrobial resistance in both healthy and ill animals, with the aim of assessing the state of veterinary antimicrobial resistance in Spain (4-6). Escherichia coli is used as a model bacterium for the analysis of isolates from sick animals. Veterinary diagnostic laboratories from official and private sectors provide the isolates, following a classic passive monitoring system with a centralized analytic approach. To avoid bias, a single isolate per farm per year (or animal in the case of pets) is studied. The identity of the farm (or owner) is kept confidential to facilitate participation. In addition, isolates are included only if their antimicrobial susceptibility has not been determined before being sent to the network. Although formal sampling is not performed, involvement of several laboratories throughout the country elicits an accurate estimate of antimicrobial susceptibility in Spain (4-6). From 1996 to 2003, the number of isolates tested was >2,300 for E. coli and 700 for Staphylococcus aureus.

The Study

E. coli MUR Mur (mr), Hung., Slovenian, and Croatian Mura (m`rä), river, c. 050 from the feces of a diarrheic pig was isolated in 2002. Routine antimicrobial susceptibility testing showed unusual resistance to amikacin. The isolate was also resistant to other aminoglycosides (streptomycin, gentamicin gentamicin /gen·ta·mi·cin/ (jen?tah-mi´sin) an aminoglycoside antibiotic complex isolated from bacteria of the genus Micromonospora, , and neomycin neomycin (nē'ōmī`sĭn), broad spectrum antibiotic effective against both gram positive and gram negative bacteria (see Gram's stain). , but not apramycin), as well as to members of other classes (sulfonamides Sulfonamides Definition

Sulfonamides are medicines that prevent the growth of bacteria in the body.
Purpose

Sulfonamides are used to treat many kinds of infections caused by bacteria and certain other microorganisms.
, trimethoprim, quinolones). Because of this unusual phenotype, the standard identification procedure (Gram staining, oxidase, indol, methyl red, citrate, and Vogues-Proskauer) (6) was confirmed by partial sequencing of the ribosomal 16S rRNA gene (data not shown) (7). Additional study of antimicrobial susceptibility by Etest (AB Biodisk, Solna, Sweden) indicated that the strain was also highly resistant to the 4,6-disubstituted deoxystreptamines netilmicin and tobramycin tobramycin /to·bra·my·cin/ (to?brah-mi´sin) an aminoglycoside antibiotic derived from a complex produced by Streptomyces tenebrarius,  (Table). We initiated an investigation to find the molecular determinants of this pattern. Plasmid DNA purified from MUR050 (Qiagen, Inc., Chatworth, California, USA) was used to transform E. coli INV INV
abbr.
in vitro fertilization
[alpha]F' (Invitrogen, Paisley, United Kingdom) (8) to yield strain INV[alpha]F' (pMUR050), which was highly resistant to aminoglycosides and sulfonamides. Resistance to aminoglycosides and the sulfonamides could be transferred from MUR050 to other E. coli by conjugation conjugation, in genetics
conjugation, in genetics: see recombination.
conjugation, in grammar
conjugation: see inflection.
 at a frequency of [approximately equal to] 1 x [10.sup.-4] per donor colony-forming unit (9).

Search for antimicrobial resistance determinants by 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) 
) using plasmid pMUR050 DNA DNA: see nucleic acid.
DNA
 or deoxyribonucleic acid

One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes.
 as template indicated the presence of genes sull n. 1. A plow.  for resistance to sulfonamides, ant3"9 for resistance to streptomycin-spectinomycin, and aph3'-I for resistance to kanamycin kanamycin /kan·a·my·cin/ (kan?ah-mi´sin) an aminoglycoside antibiotic derived from Streptomyces kanamyceticus, effective against aerobic gram-negative bacilli and some gram-positive bacteria, including mycobacteria; used as the  and neomycin. Plasmid pMUR050 DNA was digested with PstI and ligated with pUC18 DNA; the ligation mixture was used to transform INV[alpha]F' cells that were plated on agar containing ampicillin ampicillin (ăm'pĭsĭl`ĭn), a penicillin-type antibiotic that is effective against both gram-negative microorganisms and gram-positive microorganisms such as Escherichia coli.  (50 [micro]g/mL) and tobramycin (10 [micro]g/mL). The resulting colonies harbored plasmid p18MUR050 with an insert of [approximately equal to] 9 kb, which when sequenced showed armA, the structural gene for a 16S rRNA methylase (10). To confirm that armA was responsible for aminoglycoside aminoglycoside /ami·no·gly·co·side/ (-gli´ko-sid) any of a group of antibacterial antibiotics (e.g., streptomycin, gentamicin) derived from various species of Streptomyces  resistance in this strain, 2 oligonucleotides, armF (5'-GGTGCGAAAACAGTCGTAGT-3') and armR (5'-TCCTCAAAATATCCTCTATGT-3'), were used to amplify armA, which was purified, ligated into pUC 18, and transformed into E. coli INV[alpha]F' with selection on ampicillin and tobramycin. Transformant INV[alpha]F' (parmA) was highly resistant to 4,6-disubstituted deoxystreptamines (Table), which demonstrated that armA was responsible for high-level aminoglycoside resistance in MUR050.

Until now, the armA gene has been found only on conjugative plasmids of the IncL/M incompatibility group and associated with the [bla.sub.CTX-M3] gene coding for a broad-spectrum [beta]-lactamase (11). Plasmid pMUR050, however, did not confer resistance to the 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 did not belong to the IncL/M family, as tested by PCR (data not shown), which indicated that armA was carried by a different replicon rep·li·con
n.
A genetic element that undergoes replication as an autonomous unit.
. Plasmid pMUR050 DNA was digested with Sau3A, and the resulting fragments were ligated with plasmid pBluescript KS+ DNA (Stratagene, La Jolla, CA, USA). The ligation mixture was transformed into E. coli INV[alpha]F' and plated on agar containing ampicillin (50 [micro]g/mL). The inserts of plasmids from 96 random clones were sequenced with standard oligonucleotides with an ABI Abi (ā`bī) [short for Abijah], in the Bible, King Hezekiah's mother.


(Application Binary Interface) A specification for a specific hardware platform combined with the operating system.
 Prism DNA Sequencer apparatus (Perkin-Elmer, Foster City, CA, USA). Approximately half of the sequence had a high degree of identity (80%-100%) with genes of conjugative plasmid R46, which belongs to the IncN incompatibility group (12). Among them, sequences involved in plasmid replication and mobilization, such as repA and oriT, were identified (data not shown), which confirmed that armA was carried by an R46-like replicon. This finding suggests that armA could be part of a mobile genetic element that has translocated between plasmids of diverse evolutionary origins. Upstream from armA an open reading frame (ORF) has 69% identity with that of a transposase from Listonella anguillarum (GenBank accession no. AAO AAO American Association of Orthodontists; American Academy of Ophthalmology; American Academy of Otolaryngology; American Academy of Osteopathy.
AAO 
92373); this ORF and armA are flanked by 16-bp directly repeated sequences with a single mismatch (5'aggtttccactacagt-3') (GenBank accession no. AY522431). However, since aminoglycosides are rarely used in porcine production in Spain (13), and no veterinary drugs containing amikacin are registered, the sull and armA genes were conceivably co-selected by the use of sulfonamides. These antimicrobial agents are not among the 4 antimicrobial agents authorized in Europe for growth promotion (14) but are used orally to treat bacterial infections such as group E streptococcal streptococcal /strep·to·coc·cal/ (-kok´al) pertaining to or caused by a streptococcus.
Streptococcal (Streptococcus)
Pertaining to any of the Streptococcus bacteria.
 pneumonia or atrophic rhinitis and diarrhea (2).

Conclusions

These data support the notion that armA is disseminated both by conjugation and transposition, which makes further spread of armA likely. The complete sequence of plasmid pMUR050 is being determined and should help in elucidating the genetic basis for dissemination of armA.

The origin of the armA gene is still unknown. Aminoglycoside-producing environmental bacteria possess 16S rRNA methylases to protect themselves from the antimicrobial agent that they produce (15). In addition to ArmA (11), 2 other 16S rRNA methylases have been reported recently in human pathogens from Japan: RmtA in Pseudomonas aeruginosa (16) and RmtB in Serratia marcescens (17). One could speculate that actinomycetes Actinomycetes

A heterogeneous collection of bacteria that form branching filaments. The actinomycetes encompass two different groups of filamentous bacteria: the actinomycetes per se and the nocardia/streptomycete complex.
 have transferred the methylase genes to pathogenic bacteria in the environment that further spread on diverse replicons in various hosts. However, the degree of identity of armA with the structural genes for the 16S rRNA methylases in actinomycetes is <30%, which is not compatible with a recent transfer event. Alternatively, armA could have originated from a yet unknown aminoglycoside-producing actinomycetes (10).

In 2003, armA was identified as a new determinant that conferred high-level resistance to aminoglycosides in a French clinical isolate of Klebsiella pneumoniae (10) and was subsequently detected in human isolates of Enterobacteriaceae from various countries in Europe (11). The finding of armA in animal isolates shows that veterinary monitoring networks should include last-resort antimicrobial agents of high therapeutic value for humans, which could help in detecting early subtle shifts in antimicrobial resistance. In fact, armA was detected in an animal bacterium because amikacin was included in the standard antimicrobial panel in the Spanish Veterinary Antimicrobial Resistance Surveillance Network. Our finding of armA in a bacterium of animal origin highlights the importance of coordinated surveillance of human and animal isolates (18-20).
Table. Minimum inhibitory concentrations (MICs) of various
antimicrobial agents for Escherichia coli MURO50 and
E. coli with and without armA

                                  MIC ([micro]g/mL)

E. coli                    Amikacin   Tobramycin   Netilmicin

MUR050                       >256        >256         >256
INV[alpha]F'                 0.5         0.5          0.5
INV[alpha]F' (pMUR050)       >256        >256         >256
INV[alpha]F' (p18MUR050)     >256        >256         >256
INV[alpha]F' (parmA)         >256        >256         >256

                                  MIC ([micro]g/mL)

E. coli                    Gentamicin   Ciprofloxacin

MUR050                        >256           >32
INV[alpha]F'                  0.75          0.002
INV[alpha]F' (pMUR050)        >256          0.002
INV[alpha]F' (p18MUR050)      >256          0.002
INV[alpha]F' (parmA)          >256          0.002

* MICs were determined by Etest (AB Biodisk).


Acknowledgments

We thank M. Galimand, T. Lambert, and J.F. Fernandez-Garayzabal for helpful discussions.

This work was supported in part by grant AGL (programming) AGL - (Atelier de Genie Logiciel) French for IPSE. 2002/02637.

References

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(3.) Davies J, Wright GD. Bacterial resistance to aminoglycoside antibiotics. Trends Microbiol. 1997;5:234-40.

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Any of the rod-shaped, usually gram-positive bacteria (see gram stain) that make up the genus Clostridium. They are found in soil, water, and the intestinal tracts of humans and other animals. Some species grow only in the complete absence of oxygen.
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methylation
(meth´
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(11.) Galimand M, Sabtcheva S, Kantardjiev T, Poirel L, Arlet G, Courvalin P, et al. The armA aminoglycoside resistance methylase is disseminated in Enterobacteriaceae by an IncL/M plasmid mediating CTX-M3 [beta]-lactamase. In: Proceedings of the 43th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy Antimicrobial Agents and Chemotherapy (print-ISSN 0066-4804, CODEN AMACCQ; canceled ISSN 0074-9923, canceled CODEN AACHAX) is an academic journal published by the American Society for Microbiology.  (ICAAC ICAAC Interscience Conference on Antimicrobial Agents and Chemotherapy
ICAAC Iowa Community College Athletic Conference
) 2003; Sept 14-17, McCormick Place, Chicago, IL, USA.

(12.) Brown AMC, Willetts NSA. Physical and genetic map of the IncN plasmid R46. Plasmid. 1981;5:188-201.

(13.) Mateu E, Martin M. Antimicrobial resistance in enteric porcine Escherichia coli strains in Spain. Vet Rec. 2000;146:703-5.

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(16.) Yokoyama K, Doi Y, Yamane K, Kurokawa H, Shibata N, Shibayama K, et al. Acquisition of 16S rRNA methylase gene in Pseudomonas aeruginosa. Lancet. 2003;362:1888-93.

(17.) Doi Y, Yokoyama K, Yamane K, Wachino J, Shibata N, Yagi ya·gi  
n. pl. ya·gis
A directional radio and television antenna consisting of a horizontal conductor with several insulated dipoles parallel to and in the plane of the conductor.
 T, et al. Plasmid-mediated 16S rRNA methylase in Serratia marcescens conferring high-level resistance to aminoglycosides. Antimicrob Agents Chemother. 2004;48:491-6.

(18.) 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. . National Antimicrobial Resistance Monitoring System for Enteric Bacteria (NARMS NARMS National Antimicrobial Resistance Monitoring System
NARMS National Association of Rug Makers and Sculptors
): 2001 annual report. Atlanta: The Centers; 2003.

(19.) European Union Conference. The microbial threat: the Copenhagen recommendations. Copenhagen: Danish Ministry of Health and Ministry of Food, Agriculture and Fisheries; 1998.

(20.) World Health Organization. Global strategy for containment of antimicrobial resistance. Geneva Geneva, canton and city, Switzerland
Geneva (jənē`və), Fr. Genève, canton (1990 pop. 373,019), 109 sq mi (282 sq km), SW Switzerland, surrounding the southwest tip of the Lake of Geneva.
: The Organization; 2001.

Bruno Gonzalez-Zorn, * Tirushet Teshager, * Maria Casas, ([dagger]) Maria C. Porrero, * Miguel A. Moreno, * Patrice Courvalin, ([double dagger]) and Lucas Dominguez *

* Universidad Complutense de Madrid, Madrid, Spain; ([dagger]) Laboratorios Ovis, Barcelona, Spain: and ([double dagger]) Institut Pasteur, Paris, France

Dr. Gonzalez-Zorn is a researcher in the Department of Animal Health from the Veterinary School at the Universidad Complutense de Madrid, Madrid, Spain. His research interest focuses on bacterial pathogenesis and antimicrobial resistance mechanisms.

Address for correspondence: Miguel A. Moreno, Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040, Madrid, Spain; fax: 34-91-394-3908; email: mamoreno@ vet.ucm.es
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Title Annotation:DISPATCHES
Author:Dominguez, Lucas
Publication:Emerging Infectious Diseases
Geographic Code:4EUSP
Date:Jun 1, 2005
Words:1991
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