Detection of extended-spectrum beta-lactamase in Pseudomonas aeruginosa

PURPOSE: The present study was designed to detect the extended-spectrum beta-lactamase (ESBL) production in Pseudomonas aeruginosa and to evaluate the susceptibility pattern. MATERIALS AND METHODS: One hundred forty-eight isolates of P. aeruginosa were analyzed for the presence of ESBL enzyme by double disc synergy test. Antibiotic sensitivity pattern of ESBL-positive P. aeruginosa was determined. RESULTS: Of the 148 isolates tested, 30 (20.27%) were found to be positive. Maximum ESBL production was found in sputum and tracheostomy swabs (28.57%), followed by pus (24.13%), urine (19.04%), cerebrospinal fluid (CSF) and other sterile body fluids (15.38%) and blood (7.14%). All the ESBL-producing P. aeruginosa isolates were multi-drug-resistant. Isolates were 100% sensitive to imipenem. Ofloxacin was the second most (70%) effective drug. CONCLUSION: From this study, we conclude the presence of ESBL-positive P. aeruginosa in our hospital. This has important implications as carbapenems remain the only choice of treatment for infections caused by these organisms. The control measures include judicious use of antibiotics and implementation of appropriate infection control measures to control the spread of these strains in the hospital.     

Dynamics of a nosocomial outbreak of multidrug-resistant Pseudomonas aeruginosa producing the PER-1 extended-spectrum beta-lactamase

From November 1998 to August 1999, a large outbreak occurred in the general intensive care unit of the Ospedale di Circolo in Varese (Italy), caused by Pseudomonas aeruginosa producing the PER-1 extended-spectrum beta-lactamase. A total of 108 clinical isolates of P. aeruginosa resistant to broad-spectrum cephalosporins were recovered from 18 patients. Epidemic isolates were characterized by synergy between clavulanic acid and ceftazidime, cefepime, and aztreonam. Isoelectric focusing of crude bacterial extracts detected two nitrocefin-positive bands with pI values of 8.0 and 5.3. PCR amplification and characterization of the amplicons by restriction analysis and direct sequencing indicated that the epidemic isolates carried a bla(PER-1) determinant. The outbreak was of clonal origin as shown by pulsed-field gel electrophoresis analysis. This technique also indicated that the epidemic strain was not related to three other PER-1-positive isolates obtained at the same hospital in 1997. Typing by enterobacterial repetitive intergenic consensus-PCR showed that minor genetic variations occurred during the outbreak. The epidemic strain was characterized by a multiple-drug-resistance phenotype that remained unchanged over the outbreak, including extended-spectrum cephalosporins, monobactams, aminoglycosides, and fluoroquinolones. Isolation of infected patients and appropriate carbapenem therapy were successful in ending the outbreak. Our report indicates that the bla(PER-1) resistance determinant may become an emerging therapeutic problem in Europe.     

Multifocal detection of multidrug-resistant Pseudomonas aeruginosa producing the PER-1 extended-spectrum beta-lactamase in Northern Italy

Forty-four nonreplicate clinical isolates of Pseudomonas aeruginosa that were resistant to extended-spectrum cephalosporins (ceftazidime and cefepime) and aztreonam, that putatively produced an acquired extended- spectrum beta-lactamase (ESBL), according to the results of a double-disk synergy test, and that had been involved in nosocomial outbreaks were obtained from six different hospitals in northern Italy and screened for the presence of bla(PER) ESBL determinants. Twenty isolates, associated with nine independent outbreaks that occurred in five hospitals in the Milan area and its surroundings during 1995-2000, were found to carry an acquired bla(PER-1) gene. PER-1 producers representative of the nine outbreaks exhibited a multidrug resistance (MDR) phenotype, including resistance to extended-spectrum cephalosporins, aztreonam, meropenem, aminoglycosides, and in most cases, imipenem and ciprofloxacin. An analysis of macrorestriction profiles of their genomic DNAs by pulsed-field gel electrophoresis revealed an overall clonal diversity of the PER-1 producers, although interhospital clonal spread was also observed. The bla(PER-1) gene was not transferable and appeared to be chromosomally located. An analysis of the EcoRI and EcoRV restriction fragment length polymorphisms of the bla(PER-1) locus revealed identical patterns for all isolates, and the characterization of a 1.9-kb region containing bla(PER-1) revealed a conserved structure in representatives of the various clonal lineages. The present findings indicate that MDR P. aeruginosa clones producing the PER-1 ESBL are endemic to this area of northern Italy, where they have been circulating since the mid-1990s and have been associated with several nosocomial outbreaks.     

多重耐药铜绿假单胞菌超广谱β-内酰胺酶分析

目的 对多重耐药铜绿假单胞菌产β－内酰胺酶进行分析。方法 用E－试验和三相水解试验分析46株常规药敏试验全部耐药的铜绿假单胞菌的耐药表型，并用PCR扩增和产物测序方法检测其中可能产β－内酰胺酶的情况。结果 46株中8株产超广谱β－内酰胺酶，经分子生物学方法证实有blaVEB-1基因，同时还有D类酶blaOXA-10基因；46株中5株为持续高产AmpC酶；1株产生1种既能水解亚胺培南，又能被氯唑西林抑制的酶。结论 我院多重耐药铜绿假单胞菌所产β－内酰胺酶以超广谱β－内酰胺酶（8／46同时有blaVEB-1基因和blaOXA-10基因）和持续高产AmpC酶（5／46）为主。     

Clinical importance of extended-spectrum beta-lactamase (PER-1-type)-producing Acinetobacter spp. and Pseudomonas aeruginosa strains.

Recently, an extended-spectrum beta-lactamase (PER-1) was found to be disseminated among Acinetobacter spp. and Pseudomonasaeruginosa isolates in Turkey. A population-based cohort study was conducted to elucidate predictive mortality factors in patients with nosocomial infections caused by Acinetobacter spp. and P. aeruginosa, with particular reference to PER-1-type extended-spectrum beta-lactamase (ESBL) production. The study group comprised 16 and 21 non-survivors and 82 and 126 survivors in cohorts infected with Acinetobacter and P. aeruginosa, respectively. In the Acinetobacter-infected cohort, nosocomial pneumonia, hypotension and infection with a PER-positive isolate were independent predictors of mortality. In the P. aeruginosa-infected cohort, impaired consciousness, a PER-positive isolate, male sex and (with a negative relative risk) urinary tract infection were independent predictors of death. This study demonstrated the relationship of PER-1-type ESBL-producing Acinetobacter spp. and P. aeruginosa with poor clinical outcome.     

一株铜绿假单胞菌中检出两种新的整合子

目的研究铜绿假单胞菌多重耐药的临床分离株RJ217中发现的两个新整合子的结构，并分析其在多重耐药性中的作用。方法用改良三相试验分析RJ217产β-内酰胺酶的情况，用常规和长片段PCR法扩增耐药基因和整合子，并对PCR产物进行序列分析。结果发现铜绿假单胞菌RJ217携带两个新的整合子，其中一个携带veb-I型超广谱β-内酰胺酶(ESBL)基因，这两个整合子的结构分别为IS10-like-veb-I-aadB-oxa10／aadA1和aadB-oxa10／aadA1。结论整合子介导的耐药基因在RJ217的多重耐药性中发挥了重要的作用。     

Strain-tailored double-disk synergy test detects extended-spectrum oxacillinases in Pseudomonas aeruginosa

The prevalence of class D extended-spectrum oxacillinases (ES-OXAs) in ceftazidime-resistant strains of Pseudomonas aeruginosa is often underestimated by double-disk synergy tests (DDST) using clavulanate. A DDST with a customized distance between a disk of ceftazidime or cefepime and inhibitors (clavulanate and imipenem) detected 14 out of 15 different ES-OXAs.     

Outbreak of Pseudomonas aeruginosa infections with PER-1 extended-spectrum beta-lactamase in Warsaw, Poland: further evidence for an international clonal complex

Forty-one Pseudomonas aeruginosa isolates with extended-spectrum beta-lactamases (ESBLs) from a hospital in Warsaw, Poland, were analyzed. Thirty-seven isolates from several wards were collected over 9 months in 2003 and 2004. The isolates were recovered from patients with multiple types of infections, mostly respiratory tract and postoperative wound infections. All 41 isolates produced the PER-1 ESBL, originally observed in Turkey but recently also identified in several countries in Europe and the Far East. The bla(PER-1) gene resided within the Tn1213 composite transposon, which was chromosomally located. Pulsed-field gel electrophoresis and multilocus sequence typing (MLST) revealed the presence of three separate clones among the isolates. Two of these, corresponding to sequence types (STs) ST244 and ST235, were responsible for parallel outbreaks. Apart from PER-1, all the isolates produced OXA-2 oxacillinase. ST235 isolates additionally expressed a novel enzyme, OXA-74, differing by one amino acid from the OXA-17 ESBL identified originally in PER-1- and OXA-2-positive P. aeruginosa isolates from Ankara, Turkey, in 1992. These earlier Ankara isolates with PER-1, OXA-2, and OXA-17 were also classified into ST235, which is a single-locus variant of two other STs, ST227 and ST230. ST227, ST230, and ST235 all correspond to the recently described clonal complex BG11, which seems to be internationally distributed, having spread in Turkey, Greece, Italy, Hungary, Poland, Sweden, and much of Russia. It is associated with various beta-lactamases, including PER-1 and VIM metalloenzymes. This work further demonstrates the value of MLST of P. aeruginosa.     

Detection of SHV-1 beta-lactamase in Pseudomonas aeruginosa strains by genetic methods

Twelve multidrug-resistant Pseudomonas aeruginosa (MDRPA) isolates were recovered over a period of two years in the National Bone Marrow Transplant Centre of Tunisia. MDRPA isolates were isolated from seven patients and from three environmental samples. Isoelectric focusing revealed pIs of 8.2, 5.5 and 7.6 in all MDRPA isolates. These strains produced the OXA-18 extended spectrum beta-lactamase and an SHV type beta-lactamase as shown by screening PCR analysis. DNA hybridization confirmed this inference, detecting bla(SHV) gene in these isolates. Pulsed-field gel electrophoresis (PFGE) defined one predominant genomic group; group A (seven isolates) and four different genotypes containing one to two isolates. Clonally related isolates were recovered from three patients and from two washbasins. Sequencing DNA of cluster representative strains identified the classical bla(SHV-1) gene. For these strains, the nucleotide sequence of the structural bla(SHV-1) gene was nearly identical to those previously described. Such enzyme has not been reported from P. aeruginosa. This is the first report of the SHV-1 penicillinase in epidemic P. aeruginosa strain.     

Flagellar preparations from Pseudomonas aeruginosa: isolation and characterization.

Flagella from various strains of Pseudomonas aeruginosa were isolated by shearing the flagella followed by differential centrifugation to obtain typical filaments as viewed through an electron microscope. Electrophoretic analysis showed a major protein band corresponding to a flagellin with molecular weight of 53,000. Among the strains tested, flagellar antigen (FAg) preparations isolated from strains 1244 and 1210 routinely gave the highest percentage of flagellin, with the least amount of protein impurities, when grown on succinate-mineral salts medium. All FAg preparations contained 3 to 10 micrograms of 2-keto-3-deoxyoctonate-positive material per mg of protein. Strain PA-103 lacked flagella and exhibited no flagellin band, and preparations from PA-103 had a relatively higher content of 2-keto-3-deoxyoctonate. The isolation of highly purified, single-banded flagellin could be accomplished by elution of the 53,000-molecular weight gel band. Amino acid analysis showed 16 amino acids, but no proline. Antisera to FAg preparations were used to demonstrate inhibition of motility of strains RM-46 and M-2. Heated RM-46 FAg antisera and PA-103 antisera did not inhibit motility.     

Isolation and characterization of a high-molecular-weight polysaccharide from the slime of Pseudomonas aeruginosa.

A procedure is described for isolating a high-molecular-weight polysaccharide (PS) from the slime of Pseudomonas aeruginosa immunotype 1. The resultant material, obtained from the void volume of a Sephadex G-100 column, was composed of carbohydrate and water. No lipopolysaccharide (LPS), 2-keto-3-deoxyoctonoate, heptose, phosphate, or protein was detectable, and nucleic acid contamination was generally below 1%. The carbohydrate composition of the PS was glucose, rhamnose, galactose, arabinose, and mannose. PS had a molecular weight of between 100,000 and 350,000 and did not disaggregate when chromatographed in the presence of sodium deoxycholate. An antigen immunologically indistinguishable from PS could be obtained from LPS by either acetic acid hydrolysis and column chromatography or by allowing solutions of LPS to stand at room temperature for 3 days. Some of this LPS-associated polysaccharide eluted as the void volume of a G-100 column but differed from PS by its lack of galactose and arabinose. LPS also contained an immunodeterminant not shared with PS that was detected by its stability to dilute alkali treatment (0.1 N NaOH, 37 degrees C, 2 h). PS was destroyed by alkali treatment. PS appeared to represent a form of LPS polysaccharide side chain that contains galactose and arabinose and is of a high molecular weight.     

Purification and characterization of exoenzyme S from Pseudomonas aeruginosa 388.

Exoenzyme S was purified > 1,500-fold from the culture supernatant fluid of Pseudomonas aeruginosa 388 at high yield without utilization of solvents or detergents. Two proteins, with apparent molecular sizes of 53 and 49 kDa, cofractionated with exoenzyme S activity. Rabbit anti-49-kDa-protein immunoglobulin G was prepared by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis-purified 49-kDa protein as immunogen. Anti-49-kDa-protein IgG inhibited the ADP-ribosyltransferase activity of purified exoenzyme S in a dose-dependent manner, which indicated a role for the 49-kDa protein in the ADP-ribosylation reaction. Analysis by ultrafiltration showed that exoenzyme S activity and the 53- and 49-kDa proteins cofractionated and that exoenzyme S was apparently > 300 kDa in size. Urea (8 M) and 1.0% Triton X-100 reversibly decreased the apparent molecular sizes of exoenzyme S activity and the 53- and 49-kDa proteins to between 30 and 100 kDa.     

Further purification and characterization of high-molecular-weight polysaccharide from Pseudomonas aeruginosa.

Previously published reports on high-molecular-weight polysaccharides from immunotype 1 and 2 of Pseudomonas aeruginosa indicated the presence of high levels of mannose in these preparations. This mannose has been found to be due to the presence of a yeastlike mannan in high-molecular-weight polysaccharide preparations. The source of the mannan was found to be the tryptic soy broth used to grow the bacteria. Mannan could be removed from the polysaccharide preparations by chromatography over columns of concanavalin A-Sepharose. The resulting polysaccharides had the same serological reactivity against rabbit antisera and the same immunogenic properties in mice as did the mannan-containing polysaccharides. Comparison of mannan-depleted polysaccharide with preparations of high-molecular-weight polysaccharide obtained from either ultrafiltered tryptic soy broth or a chemically defined medium showed that these polysaccharides were immunologically and chemically similar. Human immune responses to mannan-depleted polysaccharide from the immunotype 1 strain of P. aeruginosa were comparable with those previously seen in humans receiving mannan-containing polysaccharides. Thus, we found that P. aeruginosa high-molecular-weight polysaccharides prepared in either tryptic soy broth and then subjected to concanavalin A-Sepharose chromatography, ultrafiltered tryptic soy broth, or a chemically defined medium were immunologically and chemically comparable.     

Enzymatic characterization of 30 kDa lipase from Pseudomonas aeruginosa ATCC 27853

An extracellular lipase from Pseudomonas aeruginosa ATCC 27853 has been purified and its enzymatic characteristics were determined. According to SDS‐PAGE and gel filtration molecular mass estimated to be 30 kDa, what classified the lipase in group I.1. Although 14 lipases from P. aeruginosa with similar molecular mass are referred to date, their basic enzymatic properties have not been reported yet. To address the gap we found: the optimal temperature and pH in water solution being 50 °C and 9.3, respectively; the lipase was inhibited with Hg²⁺ ions and sodium dodecylsulphate (SDS), while non‐ionic detergent Triton X‐100 activated the enzyme; the lipase hydrolyzed more rapidly middle chain triglycerides and it was not regiospecific; the lipase demonstrated naturally occurring stability in different organic solvents with concentrations ranging from 30 to 70%, including good thermal stability in 30% organic solvent solution. Even though strain P. aeruginosa ATCC 27853 was not isolated from extreme environment it showed activity in organic solvent suggesting that this lipase is suitable for variety of applications, including reactions in water restricted medium and bioremediation of contaminations by organic solvents. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Phenotypic and genotypic characterization of Pseudomonas aeruginosa from cystic fibrosis patients

Pseudomonas aeruginosa accounts for about one half of all pulmonary infections of cystic fibrosis (CF) patients. In this study, we analyzed 135 P. aeruginosa strains isolated from the expectorations of 55 CF adult patients attending a CF referral center over a period of five years. We assessed the genotype of the strains by pulsed-field gel electrophoresis (PFGE) and analyzed some phenotypic characteristics, such as O serotype, enzyme and mucous production, antibiotics susceptibility, and motility. PFGE allowed the typification of 97.1% of strains, revealing the presence of nine different genomic patterns. The pattern indicated as B was the most frequent, whereas patterns H and I were the most uncommon. Serotyping failed to identify 37.8% of strains and 29 out of 55 patients harbored almost one non-typable (NT) strain. During the five years of the study, we observed a progressive reduction of O6 and O10 types, but an increase of the O1 type and of NT strains. Most strains produced protease, hemolysin, and gelatinase, and were mobile. Several patients harbored the same serotype or genotype in sequential isolates, though characterized by a different susceptibility to antimicrobials. We did not observe a relationship between bacterial genotype and phenotype. This could be due to the fact that PFGE is not sensitive enough to detect subtle genotypic differences. The epidemiological importance of the genotypic characterization of bacteria-colonizing CF subjects and the surveillance measures to be adopted in CF centers are briefly discussed.     

Biochemical characterization of an alkaline metallopeptidase secreted by a Pseudomonas fluorescens isolated from soil

The extracellular endopeptidase synthesized by soil bacterium Pseudomonas fluorescens was purified to homogeneity in a four‐step procedure. The enzyme was purified 45‐fold, with a 20% recovery. The endopeptidase appeared to be a monomer with a molecular mass of approx. 50 kDa. The enzyme was active in the pH range of 7 to 10. The optimal activity was detected at pH 9.0 and at 42 °C. Enzyme activity was inhibited by EDTA, EGTA and 1,10 phenanthroline, typical metalloprotease inhibitors. Ca²⁺ activated the enzyme while Zn²⁺, Co²⁺, Cd²⁺(in high concentration) strongly inhibited it. The presence of calcium ions strongly stabilized the enzyme with regard to thermal resistance. The amino acid sequence of fragments of the enzymatic protein determined by MS analysis revealed a high similarity to the sequences of other alkaline metalloendopeptidases of bacteria belonging to the genus Pseudomonas. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

OXA-type beta-lactamases among extended-spectrum cephalosporin-resistant Pseudomonas aeruginosa isolates in a university hospital in southern Taiwan.

BACKGROUND AND PURPOSE: Data on the epidemiology of OXA-type extended-spectrum beta (beta)-lactamases (ESBLs) are limited due to difficulty of identification by routine microbiology laboratories. We determined the prevalence rate of OXA-type beta-lactamases among extended-spectrum cephalosporin (ESC)-non-susceptible Pseudomonas aeruginosa isolates at a university hospital in southern Taiwan. METHODS: A total of 1,294 ESC-non-susceptible P. aeruginosa isolates collected between 1989 and 1996 (n = 42) and between December 1999 and December 2002 (n = 1,252) were analyzed by polymerase chain reaction assays with primers specific for bla(OXA) genes and isoelectric focusing. RESULTS: Forty five isolates (3.5%) were found to produce an OXA-type beta-lactamase. Overall, 2 OXA-type ESBLs, OXA-14 (n = 2) and OXA-17 (n = 35), were detected in 37 (2.9%) isolates, and the OXA-10-type narrow-spectrum beta-lactamase was found in 8 (0.6%) isolates. OXA-10 and the 2 OXA-type ESBLs were detected in 6 (14.3%) and 4 (9.5%) of 42 ESC-non-susceptible isolates collected between 1989 and 1996. OXA-10 and OXA-17 were detected in 2 (0.2%) and 33 (2.6%) of 1,252 ESC-non-susceptible isolates collected between December 1999 and December 2002. CONCLUSIONS: These data indicate that OXA-17 was the most common OXA-type ESBL and that OXA-type beta-lactamases have decreased in ESC-non-susceptible P. aeruginosa at this hospital in recent years. Pulsed-field gel electrophoresis revealed clonal diversity among the OXA-producing isolates.     

Metallo-beta-lactamase or extended-spectrum beta-lactamase: a wolf in sheep's clothing

Diagnostic algorithms in commonly used automated bacterial identification systems fail to reliably identify a metallo-beta-lactamase in the Enterobacteriaceae. Misidentification as an extended-spectrum beta-lactamase may result in inappropriate dismissal of drugs such as aztreonam in favor of carbapenems, which may in turn select for a highly carabapenem resistant phenotype.     

Purification and characterization of cytotoxin from the crude extract of Pseudomonas aeruginosa

Pseudomonas aeruginosa cytotoxin is a protein toxin exhibiting cytotoxic effects on various eukaryotic cells. The toxin was purified from a crude extract of Pseudomonas aeruginosa 158 by trypsin treatment and serial chromatography. The purified cytotoxin was electrophoresed as a single protein band on polyacrylamide gel electrophoresis (PAGE) in the presence or absence of sodium dodecyl sulfate (SDS). The molecular weight of the purified toxin was determined to be 29,000 by SDS-PAGE and gel filtration chromatography in the presence of 6 M guanidine hydrochloride; the isoelectoric point was pH 6.0. The N-terminal amino acid sequence of the purified toxin was determined. The purified toxin showed the strongest cytotoxic effect on rabbit polymorphonuclear leukocytes and diverse degrees of cytotoxic effects on various eukaryotic cells including red blood cells and cultured cells.