VEB-1-Like Extended-Spectrum [Beta]-Lactamases in Pseudomonas aeruginosa, Kuwait.Two clinical Pseudomonas aeruginosa Pseudomonas aeruginosa A normal soil inhabitant and human saprophyte that may contaminate various solutions in a hospital, causing opportunistic infection in weakened Pts Clinical Infective endocarditis in IVDAs, RTIs, UTIs, bacteremia, meningitis, 'malignant' isolates from patients in intensive care units in Kuwait were resistant to expanded-spectrum cephalosporins Cephalosporins Definition
Cephalosporins are medicines that kill bacteria or prevent their growth.
Cephalosporins are used to treat infections in different parts of the body—the ears, nose, throat, lungs, sinuses, and and showed a synergistic effect Synergistic effect
A violation of value-additivity in that the value of a combination is greater than the sum of the individual values. between ceftazidime and clavulanic acid. This is the first report of extended-spectrum enzymes from nosocomial nosocomial /noso·co·mi·al/ (nos?o-ko´me-il) pertaining to or originating in a hospital.
1. Of or relating to a hospital.
2. isolates from the Middle East.
Pseudomonas aeruginosa has an inducible, naturally occurring cephalosporinase that confers low-level resistance to aminopenicillins and narrow-spectrum cephalosporins such as cephalothin cephalothin
a first generation cephalosporin antibiotic. Sensitive organisms include many penicillin-resistant staphylococci.
cephalothin Cefalotin® Infectious disease A parenteral semisynthetic derivative of cephalosporin C, and 3 and cefoxitin (1). Resistance to extended-spectrum cephalosporins may arise from overexpression of this cephalosporinase, acquired beta-lactamases, or both (1). The acquired beta-lactamases may be either clavulanic-acid inhibited (mostly Ambler class A enzymes) or clavulanic-acid resistant (class B and class D enzymes) (2). The class A extended-spectrum beta-lactamases (ESBLs) may derive from narrow-spectrum beta-lactamases of TEM TEM
1. transmission electron microscope.
3. transmissible encephalopathy of mink. and SHV SHV Shareholder Value
SHV Standard High Volume
SHV Steenkolen Handels Vereeniging
SHV Shreveport, LA, USA - Regional Airport (Airport Code)
SHV Sport Horse Versatility
SHV Supersonic/Hypersonic Vehicle
SHV Super Hybrid Vehicle types, as extensively reported for Enterobacteriaceae and rarely for P. aeruginosa (2). Other class A enzymes reported in P. aeruginosa include PER-1, which we first identified as chromosomally located and which is widespread in P. aeruginosa isolates in Turkey (11% of the hospital isolates) (3,4). Lately, another class A ESBL ESBL Extended Spectrum Beta Lactamase
ESBL East Staffordshire Badminton League (UK) integron-located gene, [bla.sub.VEB-1], has been identified from P. aeruginosa and enterobacterial isolates from Southeast Asia (5-7).
We report on two novel VEB-1-like beta-lactamases from P. aeruginosa clinical isolates from Kuwait. This is the first report of extended-spectrum enzymes from nosocomial isolates from this part of the world.
P. aeruginosa KU-1 was isolated in January 1999 at Ibn Sina Hospital Ibn Sina Hospital is a hospital in Baghdad, Iraq.
It was originally built for and used by Saddam Hussein, his family, and the elite of Baath Party. Uday Hussein was hospitalized there after being wounded in a failed assassination attempt in the mid 90's. in Kuwait from endotracheal endotracheal /en·do·tra·che·al/ (en?do-tra´ke-al) within or through the trachea.
Within or passing through the trachea. secretions of a 1-day-old infant with respiratory tract infection Noun 1. respiratory tract infection - any infection of the respiratory tract
infection - the pathological state resulting from the invasion of the body by pathogenic microorganisms , hospitalized in the intensive care unit (ICU ICU intensive care unit.
intensive care unit
see intensive care unit.
ICU ) unit because of severe enterocolitis enterocolitis /en·tero·co·li·tis/ (-ko-li´tis) inflammation of the small intestine and colon.
antibiotic-associated enterocolitis . He was first treated with cefotaxime, amikacin, and metronidazole metronidazole /met·ro·ni·da·zole/ (-ni´dah-zol) an antiprotozoal and antibacterial effective against obligate anaerobes; used as the base or the hydrochloride salt. It is also used as a topical treatment for rosacea. . These antibiotics were discontinued, and he was given imipenem. He improved and was discharged 7 days later. As it was a single isolate of a multidrug-resistant strain, isolation precautions were carried out only in the neonatal ICU. The infant's mother could not remember whether she had received antibiotic therapy during her pregnancy (which was uneventful). She had not traveled outside Kuwait.
P. aeruginosa KU-2 was also isolated in June 1999 from the urine of a 73-year-old man admitted to the ICU of another Kuwaiti hospital, Mubarak Al-Kabeer, with ischemic Ischemic
An inadequate supply of blood to a part of the body, caused by partial or total blockage of an artery.
Mentioned in: Antiangiogenic Therapy, Subarachnoid Hemorrhage, Ventricular Fibrillation
ischemic chest pain and bronchectiasis. On day 2 of his hospitalization, fever developed, but blood and urine cultures were negative. He was treated with ceftazidime for 12 days beginning on day 2. During his hospital stay, hematuria hematuria
Blood in the urine. It usually indicates injury or disease of the kidney or another structure of the urinary system or possibly, in males, the reproductive system. It may result from infection, inflammation, tumours, kidney stones, or other disorders. developed, followed by urine retention. On day 28, pus pus, thick white or yellowish fluid that forms in areas of infection such as wounds and abscesses. It is constituted of decomposed body tissue, bacteria (or other micro-organisms that cause the infection), and certain white blood cells. from a urinary catheter infection after transurethral transurethral /trans·ure·thral/ (trans?u-re´thral) performed through the urethra.
performed through the urethra. prostatitic surgery grew P. aeruginosa KU-2 that was susceptible to norfloxacin. P. aeruginosa KU-2 was the only P. aeruginosa strain isolated from this patient's clinical specimens. He was treated with norfloxacin. Repeated urine cultures did not yield any organism, and he was discharged 15 days later. He had no history of travel outside Kuwait.
Strains from both patients were identified by using an API-20 NE system (Biomerieux, Marcy-l'Etoile, France). Preliminary antibiotic susceptibility testing by disc diffusion (5) revealed a slight synergy between ceftazidime- and clavulanic acid-containing discs for two clinical isolates, P. aeruginosa KU-1 and KU-2. Susceptibility testing of beta-lactams for P. aeruginosa KU-1 and KU-2 was then performed by a Mueller-Hinton agar dilution method (8). Both strains showed decreased susceptibility to all beta-lactams except imipenem and piperacillin/tazobactam (Table). MICs of ceftazidime were decreased by addition of clavulanic acid and tazobactam, indicating the presence of a clavulanic-acid inhibited ESBL (Table). According to antibiotic susceptibility testing by Mueller-Hinton agar disc diffusion, P. aeruginosa KU-1 and KU-2 were also resistant to aminoglycosides (amikacin, gentamicin gentamicin /gen·ta·mi·cin/ (jen?tah-mi´sin) an aminoglycoside antibiotic complex isolated from bacteria of the genus Micromonospora, , netilmicin, tobramycin tobramycin /to·bra·my·cin/ (to?brah-mi´sin) an aminoglycoside antibiotic derived from a complex produced by Streptomyces tenebrarius, ), chloramphenicol chloramphenicol (klōr'ămfĕn`əkŏl'), antibiotic effective against a wide range of gram-negative and gram-positive bacteria (see Gram's stain). It was originally isolated from a species of Streptomyces bacteria. , tetracyclines Tetracyclines Definition
Tetracyclines are medicines that kill certain infection-causing microorganisms.
Tetracyclines are called "broad-spectrum" antibiotics, because they can be used to treat a wide variety of , sulfonamides Sulfonamides Definition
Sulfonamides are medicines that prevent the growth of bacteria in the body.
Sulfonamides are used to treat many kinds of infections caused by bacteria and certain other microorganisms. , and fluoroquinolones (except for KU-2).
Table. MICs of beta-lactams for Pseudomonas aeruginosa KU-1 and KU-2 clinical isolates, P. aeruginosa PU21(pROT-1), and PU21 reference strain MICs (mg/L) P. P. P. aeruginosa P. aeruginosa aeruginosa PU21 aeruginosa Antibiotic(a) KU-1 KU-2 (pROT-1) PU21 Amoxicillin >512 >512 >512 64 Ticarcillin >512 512 512 16 Ticarcillin+CLA 64 16 16 16 Piperacillin 64 16 16 2 Piperacillin+TZB 32 8 8 1 Ceftazidime 512 512 512 0.25 Ceftazidime+CLA 16 8 8 0.25 Ceftazidime+TZB 8 4 8 0.50 Cefotaxime 512 128 128 4 Cefotaxime+CLA 64 16 16 2 Imipenem 2 2 2 0.12 Aztreonam >512 >512 512 0.5 Aztreonam+CLA 64 8 8 0.5 (a) CLA: clavulanic acid at a fixed concentration of 2 mg/L; TZB: tazobactam at a fixed concentration of 4 mg/L.
Beta-lactamase extracts from cultures of P. aeruginosa KU-1 and KU-2 were obtained (8). Isoelectric focusing analysis (8) revealed beta-lactamases with isoelectric points of 7.4 and 8-8.4, the latter likely corresponding to P. aeruginosa AmpC cephalosporinases. Whole-cell DNAs of P. aeruginosa KU-1 and KU-2 were then obtained (3). Preliminary 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.
polymerase chain reaction
Polymerase chain reaction (PCR) ) experiments were performed with DNAs of P. aeruginosa KU-1 and KU-2 as templates and primers specific for the following class A beta-lactamases: TEM, SHV, CARB (PSE-1), GES-1, PER-1, and VEB-1 (3,5,9-11). Only PCR using internal primers for [bla.sub.VEB VEB
In currencies, this is the abbreviation for the Venezuelan Bolivar.
The currency market, also known as the Foreign Exchange market, is the largest financial market in the world, with a daily average volume of over US $1 trillion. .1] gave a positive result with an identical 642-bp fragment. External [bla.sub.VEB.1] specific primers gave 1,070-bp PCR fragments with DNAs of both P. aeruginosa strains as templates that were sequenced on both strands (6). The deduced amino acid sequences, obtained over the internet (8), identified VEB-1-like sequences that shared 99% amino acid identity with VEB-1 (Figure). Compared with VEB-1, the amino acid changes in VEB-1a and VEB-1b from P. aeruginosa KU-1 and KU-2, respectively, occurred in the putative leader peptide sequence (Figure). Thus, the hydrolytic hy·drol·y·sis
Decomposition of a chemical compound by reaction with water, such as the dissociation of a dissolved salt or the catalytic conversion of starch to glucose. activity of VEB-1a and VEB-1b should be identical to that reported for VEB-1 beta-lactamase (5).
The genetic background of [bla.sub.VEB-1a] and [bla.sub.VEB-1b] was further characterized. Plasmid extraction, conjugation conjugation, in genetics
conjugation, in genetics: see recombination.
conjugation, in grammar
conjugation: see inflection. , and electroporation electroporation (i·lekˈ·trō·p·rāˑ·sh experiments were performed (8). A plasmid (pROT-1) of ca. 70 kb carrying [bla.sub.VEB-1a] gene was identified according to hybridization hybridization /hy·brid·iza·tion/ (hi?brid-i-za´shun)
1. crossbreeding; the act or process of producing hybrids.
2. molecular hybridization
3. results by using an internal PCR-obtained probe for [bla.sub.VEB-1] (5). Plasmid pROT-1 was sell conjugative from P. aeruginosa KU-1 to in vitro obtained rifampin-resistant P. aeruginosa PU21 reference strain after selection of transconjugants onto Mueller-Hinton agar plates each containing 150 [micro]g/mL rifampin rifampin (rĭfăm`pĭn), antibiotic used in the treatment of tuberculosis. It is also used to eliminate the meningococcus microorganism from carriers and to treat leprosy, or Hansen's disease. and 200 [micro]g/mL ticarcillin. As assessed by antibiotic susceptibility testing by disc diffusion, plasmid pROT-1 conferred additional resistance to gentamicin, netilmicin, sulfonamides, and tobramycin. MICs of beta-lactams for P. aeruginosa PU21 (pROT-1) mirrored those obtained for P. aeruginosa KU-1 (Table). While the plasmid location of [bla.sub.VEB.1] gene is known only in Enterobacteriaceae (5-7), its report in P. aeruginosa may signal the evolution of its spread. The [bla.sub.VEB-1b] gene was not plasmid located, but a PCR-obtained 642-bp internal probe for [bla.sub.VEB-1] hybridized at chromosomal position of whole-cell DNA DNA: see nucleic acid.
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. of P. aeruginosa KU-2.
The [bla.sub.VEB-1] gene is located on different structures of class 1 integrons (5-7). Integrons comprise two conserved regions (5'-CS and 3'-CS) flanking an internal variable region usually containing several gene cassettes (13). Integrons are in fact expression vectors for antibiotic resistance genes that are included as gene cassettes and are neighbored (13). By using primers located either in the 5'-CS sequence and the 5' end of [bla.sub.VEB-1] or in the 3' end of [bla.sub.VEB-1] and the 3'-CS sequence (5,14), PCR amplification experiments were performed with whole-cell DNAs of P. aeruginosa KU-1 and KU-2 as templates. In one case (strain KU- 1), a PCR fragment was obtained by using [bla.sub.VEB-1] and 5'-CS primers, indicating that [bla.sub.VEB-1a] was located downstream of a class 1 integrase gene. In this case, the 4-kb PCR fragment differed from those of known [bla.sub.VEB-1] containing integrons identified in Escherichia coli and P. aeruginosa isolates (5-7). Amplimers of 1 kb were obtained for both strains using [bla.sub.VEB-1] and 3'-CS primers, showing that the 3'-CS end was present in both cases and that the [bla.sub.VEB-1]-like sequences were located next to the 3'-CS end within class 1 integrons. The attC (59-be) recombination recombination, process of "shuffling" of genes by which new combinations can be generated. In recombination through sexual reproduction, the offspring's complete set of genes differs from that of either parent, being rather a combination of genes from both parents. sites (15) located downstream of gene cassettes were identical for [bla.sub.VEB-1a] and [bla.sub.VEB.1b] to those described for [bla.sub.VEB.1] in P. aeruginosa and enterobacterial isolates identified so far from Southeast Asia (5-7). Therefore, an identical [bla.sub.VEB-1]-like gene cassette may be located on different class 1 integrons. Using 5'-CS and 3'-CS primers, two additional PCR fragments were obtained for each P. aeruginosa strain, showing that both strains contained another [bla.sub.VEB]-negative class 1 integron. For P. aeruginosa KU-1, a 950-bp PCR fragment for an aadA1a gene coding for an 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 modifying enzyme was found to be plasmid- and integron-located in Salmonella enterica serotype serotype /se·ro·type/ (ser´o-tip) the type of a microorganism determined by its constituent antigens; a taxonomic subdivision based thereon.
v. Typhimurium (16). For P. aeruginosa KU-2, a 500-bp PCR fragment encoding a putative 95 amino acid protein of unknown function was PCR amplified. It shared 71% amino acid identity with an amino acid sequence from a gene that was Tn1696 transposon-located and In4 integron-located in P. aeruginosa (17).
Finally, P. aeruginosa KU-1 and KU-2 isolates containing VEB-1-like beta-lactamases were compared with VEB-1 positive P. aerugionosa strain JES (Job Entry Subsystem) Software that provides batch communications for IBM's MVS operating system. It accepts data from remote batch terminals, executes them on a priority basis and transmits the results back to the terminals. The JES counterpart in VM is RSCS. from Thailand by using random amplified polymorphic DNA technique (10,18). The isolates were not clonally related (data not shown). Although the patients had not traveled outside Kuwait, introduction of P. aeruginosa into Kuwaiti hospitals by travelers or patients from Southeast Asia cannot be ruled out.
The presence of clavulanic-acid inhibited ESBLs in P. aeruginosa isolates may account for part of the 50% resistance to ceftazidime of P. aeruginosa strains isolates from ICUs in Kuwait (19). ESBLs in P. aeruginosa in Kuwait and other Middle Eastern hospitals may be underestimated because routine detection with a double disc synergy test may be difficult. Identification of ESBLs is of interest since they confer resistance to all extended-spectrum cephalosporins and aztreonam, whatever their MICs. This has been confirmed by experimental data using a model of pneumonia in rats with the Ambler class A ESBL, PER-1 (20).
This work underscores that very similar ESBLs may be identified in different parts of the world. It is the first report of ESBL genes characterized from P. aeruginosa isolates from the Middle East.
This work was supported by a grant from the Ministeres de l'Education Nationale et de la Recherche (UPRES, grant JE-2227), Universite Paris XI, Paris, France.
Dr. Poirel is a researcher at Hopital de Bicetre, Le Kremlin-Bicetre, France. He studies the biochemical and genetic mechanisms in beta-lactam resistance.
(1.) Chen HY, Yuan M, Livermore DM. Mechanisms of resistance to [Beta]-lactam antibiotics amongst Pseudomonas aeruginosa isolates collected in the UK in 1993. J Med Microbiol 1995;43:300-9.
(2.) Nordmann P, Guibert M. Extended-spectrum [Beta]-lactamases in Pseudomonas aeruginosa. J Antimicrob Chemother 1998;42:128-31.
(3.) Nordmann P, Naas T. Sequence analysis of PER-1 extended-spectrum [Beta]-lactamase from Pseudomonas aeruginosa and comparison with class A [Beta]-lactamases. Antimicrob Agents Chemother 1994;38:104-14.
(4.) Vahaboglu H, Ozturk R, Aygun G, Coskunkan F, Yaman A, Kaygusuz A, et al. Widespread detection of PER-type extended-spectrum [Beta]-lactamases among nosocomial Acinetobacter and Pseudomonas aeruginosa isolates in Turkey: a nationwide multicenter study. Antimicrob Agents Chemother 1997;41:2265-9.
(5.) Poirel L, Naas T, Guibert M, Chaibi EB, Labia R, Nordmann P. Molecular and biochemical characterization of VEB-1, a novel class A extended-spectrum B-lactamase encoded by an Escherichia coli integron gene. Antimicrob Agents Chemother 1999;43:573-81.
(6.) Naas T, Poirel L, Karim A, Nordmann P. Molecular characterization of In50, a class 1 integron encoding the gene for the extended-spectrum [Beta]-lactamase VEB-1 in Pseudomonas aeruginosa. FEMS Microbiol Lett 1999;176:411-9.
(7.) Tribuddharat C, Fennewald M. Integron-mediated rifampin resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother 1999;43:960-2.
(8.) Poirel L, Naas T, Nicolas D, Collet L, Bellais S, Cavallo JD, et al. Characterization of VIM-2, a carbapenem-hydrolyzing metallo-[Beta]-lactamase and its plasmid- and integron-borne gene from a Pseudomonas aeruginosa clinical isolate in France. Antimicrob Agents Chemother 2000;44:891-7.
(9.) Mercier J, Levesque RC. Cloning of SHV-2, OHIO-1 and OXA-6 [Beta]-lactamase and cloning and sequencing of SHV-1 [Beta]-lactamase. Antimicrob Agents Chemother 1990;34:1577-83.
(10.) Poirel L, Guibert M, Bellais S, Naas T, Nordmann P. Integron-and carbenicillinase-mediated reduced susceptibility to amoxicillin-clavulanic-acid in isolates of multidrug-resistant Salmonella enterica serotype typhimurium DT104 from French patients. Antimicrob Agents Chemother 1999;43:1098-2004.
(11.) Poirel L, Le Thomas I, Naas T, Karim A, Nordmann P. Biochemical-sequence analyses of GES-1, a novel class A extended-spectrum [Beta]-lactamase, and the class 1 integron In52 from Klebsiella pneumoniae. Antimicrob Agents Chemother 2000;44:622-32.
(12.) Ambler RP. The structure of B-lactamases. Philos Trans R Soc Lond B Biol Sci 1980;289:321-31.
(13.) Fluit AC, Schmitz FJ. Class 1 integrons, gene cassettes, mobility, and epidemiology. Eur J Clin Microbiol Infect Dis 1999;18:761-70.
(14.) Levesque C, Piche L, Larose C, Roy PH. PCR mapping of integrons reveals several novel combinations of resistance genes. Antimicrob Agents Chemother 1995;39:185-91.
(15.) Stokes HW, O'Gorman DB, Recchia GD, Parsekhian M, Hall RM. Structure and function of 59-base element recombination sites associated with mobile gene cassettes. Mol Microbiol 1997;26:731-45.
(16.) Tosini F, Visca P, Luzzi I, Dionisi AM, Pezella C, Petrucca A, et al. Class 1 integron-borne multiple-antibiotic resistance carried by IncFI and IncL/M plasmids in Salmonella enterica serotype typhimurium. Antimicrob Agents Chemother 1998;42:3053-8.
(17.) Hall RM, Brown HJ, Brookes DE, Stokes HW. Integrons found in different locations have identical 5' ends but variable 3' ends. J Bacteriol 1994;176:6286-94.
(18.) Williams JG, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res 1990;18:6531-5.
(19.) Jamal WY, El Din K, Rotimi VO, Chugh TD. An analysis of hospital-acquired bacteraemia bacteraemia
see bacteremia. in intensive care unit patients in a university hospital in Kuwait. J Hosp Infect 1999;43:49-56.
(20.) Mimoz O, Elhelali N, Leotard S, Jacolot A, Laurent F, Samii K, et al. Treatment of experimental pneumonia in rats caused by a PER-1 extended-spectrum [Beta]-lactamase-producing strain of Pseudomonas aeruginosa. J Antimicrob Chemother 1999;44:91-7.
Laurent Poirel,(*) Vincent O. Rotimi,([dagger]) Eiman M. Mokaddas,([dagger]) Amal Karim,(*) and Patrice Nordmann(*)
(*) Faculte de Medecine Paris-Sud, Le Kremlin-Bicetre, France; ([dagger]) Kuwait University, Kuwait
Address for correspondence: Patrice Nordmann, Service de Bacteriologie-Virologie, Hopital de Bicetre, 78 rue du General Leclerc, Le Kremlin-Bicetre 94275, France; fax: 33 1 45 21 63 40; e-mail: email@example.com