Class 1 integrons in Pseudomonas aeruginosa isolates from clinical settings in Amazon region, Brazil

A hundred and six Pseudomonas aeruginosa isolates from clinical cases were screened using PCR for the presence of integrons and associated resistance gene cassettes. Forty-four isolates harboured class 1 integrons (41.5%), of which 29 isolates (66%) also carried gene cassettes. The aacA gene was most frequently found within class 1 integrons (69%), followed by blaOXA family genes (52%). From class 1 integron-positive strains, we detected a total of 15 isolates (34%) carrying no gene cassettes. Restriction fragment-length polymorphism analysis of the integrons variable region revealed some identical structures, as well as distinct profiles indicating heterogeneity among these cassette regions. Multiresistance was observed in 71% of isolates, nevertheless no strong correlation was observed between integron presence and multiresistance. This is the first report showing class 1 integron prevalence and gene cassette content in P. aeruginosa isolates from clinical settings in the Brazilian Amazon.     

Detection of Pseudomonas aeruginosa carried a new array of gene cassettes within class 1 integron isolated from a teaching hospital in Nanjing, China

We report here novel array of gene cassettes found in single variable region of class 1 integron disseminated in Pseudomonas aeruginosa isolated from a teaching hospital in Nanjing, Jiangsu Province, China. 29 of 47 (61%) P. aeruginosa strains were confirmed haboured class 1 integron, and all the positive strains have the same variable region confirmed by PCR and RFLP methods. The variable region contained an unreported order of four gene cassettes aac(6′)-II-aadA13-cmlA8-oxa-10. Of those, cmlA8 gene was a variant of cmlA5 encoding non-enzymatic protein which putatively confer resistance to chloramphenicol. Susceptibility testing revealed multidrug-resistant mechanisms were involved in the class 1 integron positive clinical isolates. And the class 1 integron located on an about 15 kb transferable plasmid was certified by conjugation experiment and plasmid DNA analysis. The macro restriction profile indicated those clinical strains were clonally related. These authors contributed equally to this work.     

Characterization and quantification of class 1 integrons and associated gene cassettes in sewage treatment plants

Three hydrogen-based membrane biofilm reactors (MBfR) biologically reduced nitrate and perchlorate in a synthetic ion-exchange (IX) brine. Inocula from different natural saline environments were employed to initiate the three MBfRs. Under high-salinity (3%) conditions, bioreduction of nitrate and perchlorate occurred simultaneously, and the three MBfRs from the different inocula exhibited similar removal fluxes for nitrate and perchlorate. Clone libraries were generated from samples of the biofilms in the three MBfRs and compared to those of their inocula. When H₂ was the sole exogenous electron donor under high-salinity conditions, MBfR-driven community shifts were observed with a similar pattern regardless of inoculum. The following 16S rRNA gene phylogenetic analysis showed the presence of novel perchlorate-reducing bacteria in the salt-tolerant mBfR communities. These findings suggest that autohydrogenotrophic and high-salinity conditions provided strong selective pressure for novel perchlorate-reducing populations in the mBfRs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evidence for dynamic exchange of qac gene cassettes between class 1 integrons and other integrons in freshwater biofilms

Class 1 integrons carried by pathogens have acquired over 100 different gene cassettes encoding resistance to antimicrobial compounds, helping to generate a crisis in the management of infectious disease. It is presumed that these cassettes originated from environmental bacteria, but exchange of gene cassettes has surprisingly never been demonstrated outside laboratory or clinical contexts. We aimed to identify a natural environment where such exchanges might occur, and determine the phylogenetic range of participating integrons. Here we examine freshwater biofilms and show that families of cassettes conferring resistance to quaternary ammonium compounds ( qac ) are found on class 1 integrons identical to those from clinical contexts, on sequence variants of class 1 integrons only known from natural environments, and on other diverse classes of integrons only known from the chromosomes of soil and freshwater Proteobacteria . We conclude that gene cassettes might be readily shared between different integron classes found in environmental, commensal and pathogenic bacteria. This suggests that class 1 integrons in pathogens have access to a vast pool of gene cassettes, any of which could confer a phenotype of clinical relevance. Exploration of this resource might allow identification of resistance or virulence genes before they become part of multi-drug-resistant human pathogens.     

Characterization of the blaCARB-3 gene encoding the carbenicillinase-3 beta-lactamase of Pseudomonas aeruginosa.

We have isolated the blaCARB-3 structural gene encoding the CARB-3 carbenicillinase of Pseudomonas aeruginosa strain Cilote, tested the specificity of blaCARB-3 DNA probes and determined the nucleotide sequence of blaCARB-3. Three restriction fragment probes internal or delimiting the blaCARB-3 structural gene were hybridized with purified plasmid DNA coding for 18 other beta-lactamases (Blas). Under high-stringency conditions, only blaPSE-1, blaPSE-4, and blaCARB-4 sequences cross-hybridized with blaCARB-3. Sequencing of blaCARB-3 identified the structural gene which encodes a polypeptide product of 268 amino acids with a calculated estimated Mr of 29,246 for the mature form of the protein. Homology studies and computer analysis of primary structures confirmed that CARB-3 is a class-A Bla. The CARB-3 carbenicillinase differs from PSE-4 at two positions: Phe (PSE-4) instead of Leu188 (CARB-3), and Glu (PSE-4) instead of Ala266 (CARB-3), which changes the isoelectric value from (PSE-4) 5.4 to 5.75 (CARB-3). The possible effects of these two mutations were examined by comparisons on the 2 A crystal structure of the Staphylococcus aureus penicillinase, and they were shown to be silent substitutions causing no changes in the phenotype. The nucleic acid hybridization studies and sequence data confirmed that carbenicillinase-encoding bla genes are closely related and that blaCARB-3 is a variant of blaPSE-4.     

Fluoroquinolone susceptibility of Pseudomonas aeruginosa strains isolated from clinical materials

The goal of our research was an analysis of sensitivity of Pseudomonas aeruginosa to fluoroquinolones (SPX, PEF, UB, LOM, CIP, ENX, OFX, NOR). The sensitivity was tested by disk diffusion method, according to NCCLS standards. 120 strains isolated from hospitalized (76 strains) and outpatient clinic (44 strains) persons were tested. The highest sensitivity of strains was observed to norfloxacin (36.8% and 86.4% of strains, respectively) and ciprofloxacin (30.3% and 81.8%). None of tested microorganisms was sensitive to flumequine. Resistant strains, isolated from sick persons in outpatient clinics were fewer (13.6%) as compared to hospitalized persons (51.3%).     

Unnoticed spread of class 1 integrons in gram-positive clinical strains isolated in Guangzhou, China

A total of 46 gram-positive bacteria isolated from clinical specimens collected in China were subjected to PCR analysis with the intI1-specific primers, and the intI1-positive strains were further analyzed for their resistance gene cassette. All isolates possessed the class 1 integron in their genomes and the array of gene cassettes was dhfrXII-orfF-aadA2, which is very similar to other organisms except in one isolate carrying an additional copy of the class 1 integron containing the aadA2 gene cassette. Altogether, the results indicate that the class 1 integron is widespread in gram-positive clinical strains isolated in Guangzhou, China.     

Investigation of integrons/cassettes in antimicrobial-resistant Escherichia coli isolated from food animals in China

In this study,326 Escherichia coli isolates from food animals collected during the last four decades in China were characterized using antimicrobial susceptibility testing and screening for integrons/cassettes.Minimum inhibitory concentration(MIC) testing indicated that the antimicrobial resistance of E.coli has increased since the 1970s.The findings of this study present a warning to veterinary practitioners about the excessive use of antimicrobials,and suggest the necessity for surveillance and control of antimicrobial resistance in veterinary clinical medicine in China.     

Diversity of gene cassette promoters in class 1 integrons from wastewater environments

The diversity of gene cassette promoters in class 1 integrons was investigated in 47 strains isolated from wastewaters. The weak PcW and PcH1 variants predominated, suggesting that, similar to clinical environments, high rates of gene cassette recombination, rather than high expression of gene cassettes, have been preferentially selected in wastewaters.     

Identification and characterization of class 1 integron resistance gene cassettes among Salmonella strains isolated from healthy humans in China

Twenty-three strains of Salmonella spp. isolated from healthy humans in Guangdong, China, were examined for their susceptibility to ten common antibiotics and the presence of antibiotic resistance integrons. All the strains were resistant to at least one antibiotic, and 4 strains were positive for the intI1 gene. Polymerase chain reaction using in-F and in-B primers showed the existence of amplicons of 1,009 bp in two, 1,664 bp in one, and 1,009 bp and 1,664 bp in one of the intI1 -positive isolates, respectively. Sequence analysis revealed that the 1,009-bp amplicon harbored gene cassette aadA2, conferring resistance to spectinomycin, and the 1,664-bp amplicon harbored genes aadA5 and dfr17, conferring resistance to spectinomycin, streptomycin and trimethoprim. Meanwhile the experiments of plasmid conjugation and Southern hybridization with intI1 as the DNA probe indicated that all the integrons found in these strains were chromosomal. Because the strains carrying class 1 integrons were isolated from healthy humans, it suggests the need for all-round surveillance of the antibiotic resistance of pathogens.     

Gene cassettes from the insert region of integrons are excised as covalently closed circles

Integrons are DNA elements which generally include one or more discrete gene cassettes inserted at a specific site. We have recently proposed a model for the acquisition and dissemination of genes found in the insert region of integrons, which requires the existence of circularized gene cassettes. Evidence for the existence of covalently closed circular molecules consisting of one or more gene cassettes has now been obtained. Low levels of small molecules which hybridize to probes specific for individual gene cassettes were detected in plasmid DNA isolated from cells containing a plasmid which includes an integron fragment with three gene cassettes aacC1, orfE and aadA2. These molecules were only detected when the gene encoding the integron DNA integrase was also present and are thus products of site-specific cassette excision. The excised cassettes have been shown to be in the form of covalently closed supercoiled circles, by digestion with restriction enzymes exonuclease III and DNase I. The circular excision products detected included either one cassette, aadA2 or orfE, two cassettes, aacC1 and orfE or all three cassettes. The predicted sequence of the recombinant junction in the excised aadA2 cassette confirmed that excision was precise. The predicted unique sequences of the 59-base elements associated with individual genes in the circular cassette form were compiled, and the sequences of the seven-base core sites which flank 59-base elements are now, with few exceptions, exact inverted repeats.     

水产养殖环境耐药细菌中复合1型整合子的流行特征

【目的】了解复合1型整合子在水产养殖环境中的分布和流行特征。【方法】对108株分离自福建水产养殖场的耐药细菌,通过PCR和序列分析,检测其复合1型整合子上下游保守区和可变区的携带情况。【结果】有86株(79.6%)和47株(43.5%)分别携带1型整合子和ISCR1元件,这两种上下游保守区均携带的耐药细菌则有26株(24.1%),其中16株(14.8%)耐药细菌成功地扩增出上下游可变区,分布于8属9种。进一步对ISCR1上下游序列的拼接和分析,表明这16株细菌携带两种类型的复合1型整合子:(1)int I1-aac(6')-Ib-cr-arr-3-dfr A27-aad A16-qac E 1-sul1-ISCR1-sdr-qnr B6-qac E 1-sul1(15株);(2)int I1-aac(6')-Ib-cr-arr-3-dfr A27-aad A16-qac E 1-sul1-ISCR1-sap A-like-qnr B2-qac E 1(truncated)-sul1(1株,肺炎克雷伯菌C12),该阵列为新发现的复合1型整合子结构。【结论】复合1型整合子在水产养殖环境中并不少见,且存在于多种细菌中,但其基因阵列结构缺乏多样性。     

Antibiotic resistance in clinical strains of Pseudomonas aeruginosa isolated from 1979-1984

The levels and spectra of drug resistance were determined in 530 strains of P. aeruginosa isolated in hospitals of three cities of the USSR within 1979-1984. Their conjugative R plasmids were searched for and distribution of various type resistance determinants in the composition of these plasmids was investigated. The results were compared with the findings of analogous studies on clinical strains of P. aeruginosa isolated within 1976-1979. It was shown that there were a rise in the relative number of the strains resistant to kanamycin and a decrease in the occurrence of the P. aeruginosa strains resistant to streptomycin, tetracycline and sulfanilamides. The frequency of the kanamycin, carbenicillin and gentamicin resistance genes in the composition of the detected conjugative R plasmids increased. Hybridization of 32P-labeled probes containing various type antibiotic resistance determinants with strains of P. aeruginosa ML (PAO) containing conjugative R plasmids was indicative of wide spread of genes determining APH(3')II and APH(3") and determinants of classes A and C in the composition of the studied plasmids.     

Production and characterisation of a biosurfactant isolated from Pseudomonas aeruginosa UW-1

Pseudomonas aeruginosa UW-1 produced 17–24 g L⁻¹ rhamnolipid in vegetable oil-containing media in shake flask cultures in 13 days. In time course studies of growth and rhamnolipid production in a salts medium containing 6% canola oil, total bacterial count reached 2.6 × 10¹⁰ CFU ml⁻¹ after 48 h and a maximum rhamnolipid yield of 24.3 g L⁻¹ was obtained after 9 days. Rhamnolipid components were purified and separated by chloroform-methanol extraction and TLC chromatography. The major rhamnolipid components were characterised as L-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate and L-rhamnosyl-L-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate by nuclear magnetic resonance and mass spectrometry. The components were separated preparatively by silica gel column chromatography. The recovered monorhamnosyl fraction contained no dirhamnosyl moiety while the recovered dirhamnosyl fraction contained 5% of the monorhamnosyl moiety when analyzed by HPLC. The ratio of mono- to dirhamnosyl components produced by P. aeruginosa UW-1 was determined by HPLC to be 4 : 1 by weight. Purified mono- and dirhamnosyl components had the same CMC value of 40 μg ml⁻¹ and decreased the surface tension of water to 27.7 and 30.4 dynes cm⁻¹, respectively. Received 04 April 1997/ Accepted in revised form 15 July 1997     

Analysis of the Pseudomonas aeruginosa oprD gene from clinical and environmental isolates

Genomes are constantly evolving. Our report highlights the wide mutational diversity of clinical as well as environmental isolates, compared with the laboratory strain(s), through the systematic genetic analysis of a chromosomal porin gene (oprD) in relation to a specific antibiotic resistance. Mutational inactivation of the oprD gene is associated with carbapenem resistance in Pseudomonas aeruginosa. The sequence of the oprD gene of 55 Pseudomonas aeruginosa natural isolates obtained from across the world--from sources as diverse as patients and rhizospheres--was analysed. A microscale mosaic structure for this gene--resulting from multiple intra- and possibly interspecies recombinational events--is reported. An array of independent and seemingly fast-occurring defective oprD mutations were found, none of which had been described before. A burn wound isolate demonstrated unusually high overall sequence variability typical of mutator strains. We also present evidence for the existence of OprD homologues in other fluorescent pseudomonads.     

产ESBLs大肠埃希菌整合子及其相关基因盒的研究

目的检测产超广谱β-内酰胺酶（ESBLs）大肠埃希菌中整合子的整合酶及插入的相关基因盒情况,分析整合子对细菌耐药性的影响。方法采用K-B琼脂扩散法对45株临床分离的产ESBLs大肠埃希菌进行药敏试验;应用PCR法检测45株产ESBLs大肠埃希菌Ⅰ类、Ⅱ类和Ⅲ类整合子;对Ⅰ类整合子阳性菌进行整合子相关基因盒检测。结果45株菌中有27株（60．0％）含有Ⅰ类整合子,没有检测到Ⅱ类和Ⅲ类整合子阳性菌。在Ⅰ类整合子阳性菌中,有23株携带Ⅰ类整合子相关基因盒（85．2％）,5种不同的基因盒图谱,片段大小在600～2322bp,分离自同一科室的部分菌株携带大小相同的基因盒;Ⅰ类整合子阳性菌株的耐药率高于整合子阴性的菌株。结论Ⅰ类整合子及整合子相关基因盒在产ESBLs大肠埃希菌株中分布广泛,整合子在细菌耐药中发挥作用。     

Antibiotic susceptibility and occurrence of ESBL in Pseudomonas aeruginosa strains isolated from different clinical specimens

The aim of this study was to evaluate the drug susceptibility of 132 P. aeruginosa strains isolated from patients hospitalized in SPSK University Hospital in Bialystok. The isolates were obtained from clinical specimens over an 11-month period in 2001 and 2002. All the strains were identified in automatic ATB system using API 20 NE strips, and their susceptibility to antibiotics was tested by standard disc-diffusion method and agar dilution method. The minimal inhibitory concentration (MIC) was determined for five antibiotics: piperacillin, amikacin, ceftazidime, imipenem and ciprofloxacin. The majority of strains were susceptible to ceftazidime (91.7%), piperacillin combined with tazobactam (85.6%), amikacin (80.3%), meropenem and imipenem (81.8%). Many of our strains were resistant to cefotaxime (73.5%), ticarcillin (53%) and ciprofloxacin (48.5%). Also, the trial was undertaken to detect strains producing extended-spectrum beta-lactamases (ESBL) and inducible beta-lactamases (IBL) among P. aeruginosa rods isolated from different specimens. ESBL-producing strains were detected with double disc test (DDST) and combination double disc (CD) test. Clavulanate was applied as the inhibitor of these beta-lactamases. Strains producing ESBL were not found. On the other hand, as many as 127 P. aeruginosa strains (96.2%) produced inducible beta-lactamases (IBL).