Antibiotics resistance of Proteus mirabilis isolated from patients with acute diarrhea / 天津医药

Objective To investigate antibiotics resistance of Proteus mirabilis isolated from stools of patients with acute diarrhea for the prevention and treatment of its infection and the rational use of antibiotics. Methods Stool samples of acute diarrhea patients were collected in the diarrhea outpatient clinic of the Second Hospital of Tianjin Medical University and Tianjin Medical University General Hospital from 2013 to 2014. Enrichment culture and biochemical identification were used to isolate and identify Proteus mirabilis, which were further performed antimicrobial susceptibility testing and class 1 integron detection. Extended spectrum β-lactamases (ESBLs) phenotype and ESBLs genes (TEM, OXA and CTX-M) were amplified by polymerase chain reaction (PCR), and sequencing were carried on in parts of suspected isolates. ESBLs-positive strains were analyzed by pulsed-field gel electrophoresis (PFGE). Results A total of 277 strains of non-repetitive Proteus mirabilis were isolated, and 268 of them were performed antimicrobial susceptibility testing (the remaining 9 strains failed to recover). Relative higher resistant rates were trimethoprim/sulfamethoxazole (30.2%), ampicillin (25.4%), nalidixic acid (25.7%), streptomycin (21.6%) and chloramphenicol (21.3%). The multiple drug resistance rate was 24.6% (66/268). The positive rate of class 1 integron was 22.8%(61/268). Resistance rates to third-generation cephalosporin, ciprofloxacin and imipenem were less than 10%, but 4 isolates were resistant to imipenem, third-generation cephalosporin, fluoroquinolones, trimethoprim/sulfamethoxazole, and chloramphenicol simultaneously. Three cefotaxime-resistant strains (1062, 1505 and 1650) were positive for ESBLs phenotype and harbored CTX-M extended-spectrum β-lactamase genes, among them 2 strains also carried TEM and/or OXA β-lactamase genes. The clustering analysis of pulsed-field gel electrophoresis (PFGE) displayed that the similarities between 1505 and 1650 were 85.7%, and the similarity with 1062 was 58.1%. Conclusion Proteus mirabilis isolated from patients with acute diarrhea in our city show significant multidrug resistance, high positive rate of class 1 integron, and emergence of ESBLs-positive strains resistant to imipenem and fluoroquinolones, which pose a threat to public health. Rational use of antibiotics is important in both clinical and nonclinical settings.

Characterization of the variable region in the class 1 integron of antimicrobial-resistant Escherichia coli isolated from surface water

Abstract Fecal bacteria are considered to be a potential reservoir of antimicrobial resistance genes in the aquatic environment and could horizontally transfer these genes to autochthonous bacteria when carried on transferable and/or mobile genetic elements. Such circulation of resistance genes constitutes a latent public health hazard. The aim of this study was to characterize the variable region of the class 1 integron and relate its genetic content to resistance patterns observed in antimicrobial-resistant Escherichia coli isolated from the surface waters of Patos Lagoon, Southern Brazil. Genetic diversity of the isolates and presence of the qacEΔ1 gene, which confers resistance to quaternary ammonium compounds, were also investigated. A total of 27 isolates were analyzed. The variable region harbored dfrA17, dfrA1 and dfrA12 genes, which confer resistance to trimethoprim, and aadA1, aadA5 and aadA22 genes that encode resistance to streptomycin/spectinomycin. Most of the isolates were considered resistant to quaternary ammonium compounds and all of them carried the qacE Δ1 gene at the 3′ conserved segment of the integron. ERIC-PCR analyses of E. coli isolates that presented the integrons showed great genetic diversity, indicating diverse sources of contamination in this environment. These results suggest that fecal bacteria with class 1 integrons in aquatic environments are potentially important reservoirs of antibiotic-resistance genes and may transfer these elements to other bacteria that are capable of infecting humans.

Isolation and characterization of antimicrobial-resistant Escherichia coli from national horse racetracks and private horse-riding courses in Korea

Limited information is available regarding horse-associated antimicrobial resistant (AR) Escherichia (E.) coli. This study was designed to evaluate the frequency and characterize the pattern of AR E. coli from healthy horse-associated samples. A total of 143 E. coli (4.6%) were isolated from 3,078 samples collected from three national racetracks and 14 private horse-riding courses in Korea. Thirty of the E. coli isolates (21%) showed antimicrobial resistance to at least one antimicrobial agent, and four of the AR E. coli (13.3%) were defined as multi-drug resistance. Most of the AR E. coli harbored AR genes corresponding to their antimicrobial resistance phenotypes. Four of the AR E. coli carried class 1 integrase gene (intI1), a gene associated with multi-drug resistance. Pulsed-field gel electrophoretic analysis showed no genetic relatedness among AR E. coli isolated from different facilities; however, cross-transmissions between horses or horses and environments were detected in two facilities. Although cross-transmission of AR E. coli in horses and their environments was generally low, our study suggests a risk of transmission of AR bacteria between horses and humans. Further studies are needed to evaluate the risk of possible transmission of horse-associated AR bacteria to human communities through horse riders and horse-care workers.

Plásmido conjugativo portador de integrón clase 1 responsable de la resistencia a los antibióticos en aislados de vibrio cholerae O1 en Venezuela / Conjugative plasmid and a class 1integron responsible for the resistance to antibiotics in vibrio cholerae O1 isolates in Venezuela

Se han descrito aislados de Vibrio cholerae resistentes a una amplia variedad de antibióticos. En Venezuela, durante el brote de cólera ocurrido entre noviembre de 1998 y enero 2000 fueron reportados por primera vez aislados de V. cholerae O1 resistentes a ampicilina, trimetoprim-sulfametoxazole. Usando experimentos de conjugación se determinó la capacidad de transferir los determinantes de resistencia a ampicilina y trimetoprim-sulfametoxazole en 11 aislados. La visualización de plásmidos se realizó utilizando la digestión con nucleasa S1 y electroforesis en campo pulsante. La presencia de integrones de clase 1 fue establecida por PCR y se obtuvo la secuencia de la región variable del integrón. Los determinantes de resistencia fueron transferidos en un plásmido conjugativo de aproximadamente 170 kbp, común a todos los aislados. La resistencia a trimetoprim esta codificada en el gen dfra15, el cual se encuentra en un integrón clase 1 presente en el plásmido. En este estudio, se caracterizó la localización genética de los determinantes que codifican la resistencia a los antibióticos, y al conocer el mecanismo probable de dispersión de los determinantes de resistencia se podrán implementar medidas de control más adecuadas.

Vibrio cholerae has been reported to be resistant to a wide range of antibiotics. V. Cholerae O1 strains resistant to ampicillin, trimethoprim-sulfamethoxazole were isolated for the first time in Venezuela during a cholera outbreak that occurred between November 1998 and January 2000. Using conjugation experiments, the capacity of transfer of the resistance determinants in 11 strains resistant to ampicillin and trimethoprim-sulfamethoxazole was investigated. Plasmid analysis was done by S1 nuclease digestion and pulsed field gel electrophoresis. The presence of class 1 integrons was determined by PCR and the sequence of the gene harbored in the variable region of the integron was obtained. The antibiotic resistance determinants were transferred by a conjugative plasmid of approximately 170 kbp, common to all the isolates. Resistance to trimethoprim is encoded by the dfra15 gene that is harbored by a class 1 integron present in the plasmid. In this study, the genetic location of the determinants that code for resistance to antibiotics was characterized, and knowing the probable mechanism of dispersion of the determinants of resistance, control measures can be implemented most appropriate

The sul1 Gene in Stenotrophomonas maltophilia With High-Level Resistance to Trimethoprim/Sulfamethoxazole

Emerging resistance to trimethoprim/sulfamethoxazole (SXT) poses a serious threat to the treatment of Stenotrophomonas maltophilia infections. We determined the prevalence and molecular characteristics of acquired SXT resistance in recent clinical S. maltophilia isolates obtained from Korea. A total of 252 clinical isolates of S. maltophilia were collected from 10 university hospitals in Korea between 2009 and 2010. Antimicrobial susceptibility was determined by using the CLSI agar dilution method. The sul1, sul2, and sul3 genes, integrons, insertion sequence common region (ISCR) elements, and dfrA genes were detected using PCR. The presence of the sul1 gene and integrons was confirmed through sequence analysis. Among the 32 SXT-resistant isolates, sul1 was detected in 23 isolates (72%), all of which demonstrated high-level resistance (> or =64 mg/L) to SXT. The sul1 gene (varying in size and structure) was linked to class 1 integrons in 15 of the 23 isolates (65%) harboring this gene. None of the SXT-susceptible isolates or the SXT-resistant isolates with a minimum inhibitory concentration of 4 and 8 mg/L were positive for sul1. Moreover, the sul2, sul3, and dfrA genes or the ISCR elements were not detected. The sul1 gene may play an important role in the high-level SXT resistance observed in S. maltophilia.

Investigation of class 1 integrons in Klebsiella pneumoniae clinical and microbiota isolates belonging to different phylogenetic groups in Recife, State of Pernambuco

Introduction The high prevalence of Klebsiella pneumoniae infections is related to the ability of K. pneumoniae to acquire and disseminate exogenous genes associated with mobile elements, such as R plasmids, transposons and integrons. This study investigated the presence of class 1 integrons in clinical and microbiota isolates of K. pneumoniae belonging to different phylogenetic groups and correlated these results with the antimicrobial resistance profiles of the studied isolates. Methods Of the 51 isolates of K. pneumoniae selected for this study, 29 were from multidrug-resistant clinical isolates, and 22 were from children's microbiota. The susceptibility profile was determined using the disk diffusion method, and class 1 integrons were detected through polymerase chain reaction (PCR). Results The results showed that none of the 22 microbiota isolates carried class 1 integrons. Among the 29 clinical isolates, 19 (65.5%) contained class 1 integrons, and resistance to sulfamethoxazole/trimethoprim was identified in 18 of these isolates (94.7%). Among the K. pneumoniae isolates with class 1 integrons, 47% belonged to the KpI phylogenetic group, and one isolate (14.3%) carrying these genetic elements belonged to the KpIII group. Conclusions The wide variety of detected class 1 integrons supports the presence of high rates of antimicrobial resistance, genetic variability, and rapid dissemination of beta-lactamase genes among K. pneumoniae clinical isolates in recent years in hospitals in Recife-PE, Brazil. The findings of this study indicate that the surveillance of K. pneumoniae integrons in clinical isolates could be useful for monitoring the spread of antibiotic resistance genes in the hospital environment. .

Loop-mediated isothermal amplification assays for screening of bacterial integrons

BACKGROUND: The occurrence and prevalence of integrons in clinical microorganisms and their role played in antimicrobial resistance have been well studied recently. As screening and detection of integrons are concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for integrons detection. RESULTS: In this study, three loop-mediated isothermal amplification (LAMP) assays targeting on class 1, 2 and 3 integrons were implemented and evaluated. Optimization of these detection assays were performed, including studing on the reaction temperature, volume, time, sensitivity and specificity (both primers and targets). Application of the established LAMP assays were further verified on a total of 1082 isolates (previously identified to be 397 integron-positive and 685 integron-negative strains). According to the results, the indispensability of each primer had been confirmed and the optimal reaction temperature, volume and time were found to be 65°C, 45 min and 25 µL, respectively. As application was concerned, 361, 28 and 8 isolates carrying intI1, intI2 and intI3 yielded positive amplicons, respectively. Other 685 integron-negative bacteria were negative for the integron-screening LAMP assays, totaling the detection rate and specificity to be 100%. CONCLUSIONS: The intI1-, intI2- and intI3-LAMP assays established in this study were demonstrated to be the valid and rapid detection methodologies for the screening of bacterial integrons.

Class 1 integrons contributes to antibiotic resistance among clinical isolates of Escherichia coli producing extended-spectrum beta-lactamases.

Objectives: The objective of this study is to determine the prevalence of antibiotic resistance factors, including the production of extended-spectrum beta-lactamases (ESBLs) and the presence of class 1 integrons among Escherichia coli isolated from clinical specimens. Materials and Methods: Bacterial species identifi cation was performed using a VITEK-2 system (VITEK2 GN-card; bioMérieux, France). Antimicrobial susceptibility testing was determined using the disk diffusion method according to the 2010 Clinical and Laboratory Standards Institute guidelines. Polymerase chain reaction (PCR) was used to detect integrons and amplify variable regions of the blaTEM, blaSHV and blaCTX-M genes. Gene cassettes were detected by deoxyribonucleic acid sequencing. Results: In this study, 58% (100/172) of clinical E. coli isolates were identifi ed as ESBL producers. We found that 90% of the ESBL-producing E. coli isolates harbored the blaCTX-M gene, whereas only 59% and 32% possessed the blaTEM and blaSHV genes respectively. The presence of class 1 integrons was based on the detection of the integrase gene by PCR. A total of 69% of the ESBL-producing isolates were integron-positive. Resistance to 10 antibiotics, including quinolones, sulfonamides and -lactam/enzyme inhibitors, was signifi cantly higher in the class 1 integron-positive isolates (P < 0.05). The occurrence of class 1 integrons in blaTEM, blaSHV and blaCTX-M gene carriers was 72.9%, 84.4% and 68.9%, respectively. Class 1 integrons were detected in 61.5% of the isolates with only one ESBL genotype, but in 69.0% and 92.3% of the isolates with two or three different ESBL genotypes, respectively. Conclusions: Our fi ndings indicate that clinical strains of bacteria with multiple ESBL genotypes may have greater opportunities to carry class 1 integrons.

Sewage treatment plant serves as a hot-spot reservoir of integrons and gene cassettes.

This study investigated the occurrence and abundance of class 1 integrons and related antibiotic resistance genes (ARGs) in a sewage treatment plant (STP) of China. Totally, 189 bacterial strains were isolated from influent, activated sludge and effluent, and 40 isolates contained the integons with a complete structure. The intI1-carrying isolates were found to harbor two types of gene cassettes: dfr17-aadA5 and aadA2, conferring resistances to trimethoprim and streptomycin, which were further confirmed by antimicrobial susceptibility analysis. Many other gene cassettes were carried on integron, including qnrVC1, catB-8-blaoxa-10-aadA1-aac(6'), aadB-aacA29b, aadA2, aac(6')-1b, aadA6 and aadA12, which were detected using DNA cloning. Quantitative real time PCR showed that over 99% of the integrons was eliminated in activated sludge process, but average copy number of integrons in given bacterial cells was increased by 56% in treated sewage. Besides integrons, other mobile gene elements (MGEs) were present in the STP with high abundance. MGEs and the associated ARGs may be wide-spread in STPs, which constitute a potential hot spot for selection of antibiotic resistant bacteria and horizontal transfer of ARGs.

Molecular Characterization of Citrobacter freundii Isolated from Neonates in Neonatal Intensive Care Unit of Nepal.

Introduction: Nosocomial Citrobacter spp. is emerging as a successful nosocomial pathogen in neonates in Nepal. The important risk factor being poor infection prevention and control practices. The objective of this study was to investigate the clonal relatedness of Citrobacter freundii isolated from clinical and nonclinical sources in Neonatal Intensive Care Unit (NICU) and to determine the presence of Extended Spectrum Beta-Lactamase (ESBL) genes and class 1 integron element. Materials and Methods: Polymerase chain Reaction (PCR) and PCR-Randomly Amplified Polymorphic DNA typing of the isolates were performed in three isolates to amplify class 1 integron element integrase gene, ESBL genes, and to study the clonal relatedness, respectively. Results: Two isolates harbored class 1 integron element. The blaCTX-M was present in all isolates and blaTEM-1 was present in one isolate. An isolate carried blaCTX-M and blaTEM-1 genes. All of these isolates were not clonally related. Conclusion: The study for the first time documented the emergence and spread of ESBL genes and class 1 integron element in multidrug resistant C. freundii in Nepal and urge for monitoring and surveillance of these strains.

Analysis of Acquired Resistance Genes in Stenotrophomonas maltophilia / 대한진단검사의학회지

BACKGROUND: Stenotrophomonas maltophilia is a gram-negative bacillus and a nosocomial pathogen in immunocompromised patients. Trimethoprim/sulfamethoxazole (TMP/SMX) is the drug of choice for treating S. maltophilia infection; however, resistance to TMP/SMX is increasing. In this study, we investigated the relationship between the incidence of TMP/SMX resistance and the presence of sul genes and mobile elements. METHODS: A total of 120 S. maltophilia isolates were collected from 3 university hospitals between April 2007 and April 2009. Antimicrobial susceptibilities were determined using the disk diffusion method. PCR and DNA sequencing were conducted for the detection of sul1, sul2, class 1 integron, and ISCR2 element. Repetitive extragenic palindromic sequence-based PCR (REP-PCR) was carried out to evaluate the genetic relatedness. RESULTS: The TMP/SMX-resistant (R) isolates harbored a significantly higher proportion of sul1 gene and class 1 integron than TMP/SMX-susceptible (S) isolates (P<0.001). Seventeen of 28 isolates with sul1 also had a class 1 integron, but none of the isolates without sul1 had a class 1 integron. The identified gene cassettes within class 1 integrons include aacA4, aadA1, aac6'-II, and qac. None of the 120 isolates carried sul2, glmM, or ISCR2 element. REP-PCR did not show any genetic relatedness among the isolates. CONCLUSIONS: In Korea, the resistance of S. maltophilia isolates to TMP/SMX is due to sul1 within a class 1 integron rather than to sul2. The class 1 integron also harbors multiple antibiotic resistance genes in addition to sul1, and therefore it could mediate multidrug resistance in S. maltophilia.

Analysis of drug-resistant genes of 60 Acinetobacter baumannii isolates / 基础医学与临床

Objective To investigate β-lactamase gene and class 1 integron gene from 60 clinical Acinetobacter baumannii isolates.Methods The minimal inhibitory concentrations (MICs) for 16 antibiotics widely used were determined using the standard broth microdilution method.The β-lactamase gene,class 1 integron gene and adeB gene were determined by PCR and then sequenced.Results Fifty-three strains of the 60 A.baumanii isolates were multi-drug resistant.OXA-23 gene was detected positive in six A.baumanii isolates,which were all resistant to more than five antimicrobial agents including carbapenem and showed high resistance to many antibiotics.Thirtyeight strains earring PER-1 gene showed higher resistance to cephalosporins than those without this gene (P＜0.01).Class 1 integron gene was positive in 45 strains,which exhibited significantly higher multiple resistance than those without this gene (P＜0.01).Twenty-five strains carrying both class 1 integron and PER-1 genes had a markedly higher multiple resistance (P＜0.01),but not in resistant level,compared to the 7 strains without these two genes.Conclusion Class 1 integron and β-lactamase gene may be the causes of muhidrug-resistance of A.baumanii.The strains carrying OXA-23 gene always showed multiple and high resistance to several antibiotics,so effective measures must be taken to control the epidemic of these strains.

Characterization of Class 1 Integrons in Metallo-beta-lactamase-producing Pseudomonas aeruginosa / 대한임상미생물학회지

BACKGROUND: The genes of metallo-beta-lactamase (MBL), a powerful carbapenemase, are carried as a part of the mobile gene cassettes inserted into integrons playing an important role in rapid dissemination of antibiotic resistance genes among bacterial isolates. In this study, we investigated carbapenemase genes and class 1 integrons integrated into the gene cassettes in imipenem-non susceptible P. aeruginosa. METHODS: From July 2006 to March 2008, 81 consecutive, non-duplicate, imipenem-non susceptible P. aeruginosa were isolated at Chungnam National University Hospital in Chungcheong province of Korea. The modified Hodge and double disk synergy tests were conducted for the screening of carbapenemase and MBL production, respectively, and PCR and DNA sequencing were performed for the detection of carbapenemase genes and class 1 integron gene cassettes. We also employed the repetitive element sequence-based (Rep)-PCR method for an epidemiologic study. RESULTS: MBLs were detected in 13.6% (11/81) of imipenem-non susceptible P. aeruginosa. Ten isolates were found to carry blaIMP-1, whereas 1 isolate was found to carry a blaVIM-2. All of the IMP-1-producing strains harbored 4.0 kb class 1 integron containing chloramphenicol, aminoglycoside, and beta-lactam- resistant genes. However, blaIMP-1 was not detected at class 1 integron. A 2.5 kb class 1 integron harboring blaVIM-2 was detected in a VIIM-2- producing strain. One identical pattern was observed in ten IMP-1 producing strains. CONCLUSION: IMP-1 producing P. aeruginosa strains are currently distributed throughout Chungcheong province of Korea. In particular, all of the strains harbored class 1 integrons containing variant antibiotic resistance gene cassettes.

Characteristic of class 1 integron-cassette among Shiga toxin-producing E. coli isolates in China / 吉林大学学报(医学版)

Objective To explore the distribution and characteristic of class 1 integrons among Shiga toxin-producing E.coli(STEC) in China,and to elucidate the type of cassettes.Methods Antibiotics susceptibility was tested by the disk diffusion method,class 1 integron was detected by PCR assay and PCR products were sequenced and analyzed in eight isolates.Results Four isolates were multiple-drug resistant whose antibiogram were ampicillin,tetracycline,erythromycin,sulfamethoxazole/trimethoprim and ciprofloxacin,but two isolates conferred resistance of streptomycin and spectinomycin simultaneously.Only the two isolates carried class 1 integrons ranging in 1 000 bp which located on the plasmid of 7 800 bp.The sequenced PCR product demonstrated that the 1 000 bp integron harbored aad A1 gene cassettes conferred the resistance of streptomycin and spectinomycin;and linked with sul 1 and qacE?1 gene conferred the resistance of sulfamethoxazole and quaternary disinfectants.Conclusion The class 1 integrons exists among STEC isolates in China and determines the resistant antibiotics.

Identification and characterization of class 1 integron among E. coli from healthy students' enteric strains / 吉林大学学报(医学版)

Objective To explore the distribution and characterization of class 1 integrons in E.coli from healthy feces,and to elucidate the status of gene-cassettes.Methods Routine method was used to isolate E.coli,antibiotics susceptibility was tested by the disk diffusion method;class 1 integron was detected by PCR assay;PCR products were sequenced and analyzed.Results Of 97 samples,76 isolates were identified,and 25 isolates were multiple-drug resistant.The antibiogram was sulfamethoxazole-trimethoprim,ampicillin,streptomycin,tetracycline,erythromycin.14 of 25 isolates carried class 1 integrons,and the size of integrons differed from 1 800 bp(10 strains) to 750 bp(4 strains).The sequenced PCR product demonstrated that the 1 800 bp integron laboured aadA1-dfrA14-orf gene cassette conferred the resistance to sulfamethoxazole-timethoprim,streptomycin and aminoglycoside;the 750 bp integron laboured dfrA14 gene cassette conferred the resistance to sulfamethoxazole-trimethoprim.Conclusion The different kinds of class 1 integrons exist in E.coli from the healthy students,and determine the multiple-resistant antibiotics.

Association of class 1 integrons of Acinetobacter baumannii with resistance / 临床检验杂志

Objective To monitor the distribution of class 1 integrons of Acinetobacter baumannii and interpret the association of class 1 integrons with the resistance in clinical isolates.Methods Susceptibility to 14 different antimicrobial agents was determined by agar dilution method.DNA of all clinical isolates was extracted to detect class 1 integrons by PCR.Results The differences between integron-positive isolates and integron-negative isolates was significant in the resistance to all tested antibiotics except for Imipenem,Cefepime and Cefoperazone plus Sulbactam.Different types of class 1 integrons showed different resistance types.Conclusions There were some close relations of class 1 integrons with multi-drug resistance as well as resistance types in Acinetobacter baumannii.

Changes in patterns of antimicrobial susceptibility and class 1 integron carriage among Escherichia coli isolates

The worldwide use of antimicrobials in different fields has created enormous pressure for the selection of resistance among opportunistic bacterial pathogen. One hundred four E. coli isolates were collected and identified from swine with diarrhea in Korea during the period of 2002. The isolates showed highly resistant to streptomycin (99. 0%), tetracycline (97. 1%), neomycin (91. 3%)and carbenicillin (84. 6%)in antimicrobial susceptibility test. Moreover, all of the isolates showed multiple antimicrobial resistant to more than 3, and 85%of them were resistant to more than 7 of total 14 antimicrobial agents. In comparison with isolates in 1998, resistance to antimicrobials was more frequent among the isolates in 2002. Presence of class 1 integrons was investigated through amplification of the gene with PCR, and could be classified 8 groups by pattern of 4 different amplicons. Class 1 integrons were observed in 67 strains (64. 2%)of E. coli from swine in Korea. One and 1. 6 kbp of amplicons were revealed to contain aadA1 and aadB-aadA1 gene cassettes respectively. Two kbp of amplicon had three different gene cassettes, dhfrXII-orfF-aadA2, and 3. 0 kbp of amplicon includes aadB-cmlA1 gene cassettes.