Molecular Epidemiology And Antibiotic Resistance Of Three Gram-negative Bacteria Isolated From Nosocomial Infection Patients In Guangdong Province

BackgroundKlebsiella pneumoniae is a common bacteria in human respiratory tract and intestinal tract, is also an important opportunistic pathogen, can cause respiratory, urinary tract, intestinal tract, blood, surgical incision, and many other parts of skin and soft tissue infections. Stenotrophomonas maltophilia as opportunistic pathogens, not only widely distributed in nature, human skin, oropharynx, upper respiratory tract and intestinal also were colonized, the separation rate in the hospital environment and medical personnel is also high. In recent years, infection caused by Stenotrophomonas maltophilia rise and has become an important pathogen in hospital infections. Pseudomonas aeruginosa is a common opportunistic pathogen, widely distributed in nature, healthy skin, intestinal, respiratory and hospital wards, medical equipment, hospital-acquired infections is one of the major pathogens. Resistance drops in the body (such as infection, surgery, cancer chemotherapy, burns and other invasive medical tracheotomy operation) can cause local or systemic human infection. All three gram-negative bacteria infection in the hospital plays an increasingly important position. Therefore, the in-depth understanding of the three bacterial characteristics and molecular epidemiology of nosocomial infection and drug resistance, to prevent and control hospital infection is important.Content and objectives(1)To understand the epidemiological characteristics of nosocomial infection caused by Klebsiella pneumoniae, Stenotrophomonas maltophilia and Pseudomonas aeruginosa in 5 hosptials of Guangdong provice, and by pulsed-field gel electrophoresis (PFGE) isolated the three Gram-negative bacteria genotypes, analysis of hospital infection homologous strain. (2)To understand the antimicrobial resistance pattern of the Klebsiella pneumoniae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia in Guangdong Provine, and demonstrate the resistance rates of isolates from different sources, and the differences between the strains resistance patterns and genotypes relationship.(3) Multiple resistant strains by PCR amplification of and integrated sub-categories, to understand the carrying status of IntⅠ, IntⅡ, IntⅢof Klebsiella pneumoniae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia in Guangdong region.(4)Of integron isolate was integron RFLP analysis and sequence of inserted gene cassette, to understand the resistance genes carried by strains of the types of boxes, features, and with the strain resistance.(5) Understanding the disinfectant resistant gene qacEΔ1 in Klebsiella pneumoniae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia carrying status of Guangdong region, and qacEΔ1 positive strains resistance to disinfectants in hospitals the compared with qacEΔ1 negative strains and standard strain.Methods(1) 160 strains of Klebsiella pneumoniae,134 Stenotrophomonas maltophilia and 243 strains of Pseudomonas aeruginosa, which were isolated from nosocomial infection in 5 hospitals of Guangdong Province, all strains confirmed by biochemical identification, troubleshooting and further confirmed by the VITEK 232 automatic bacteria identification. Meanwhile, cases of hospital infection data were collected and descriptive analyzed.(2)PFGE was used to analyze the homology of 160 strains of Klebsiella pneumoniae,134 Stenotrophomonas maltophilia and 243 strains of Pseudomonas aeruginosa,which were islated from 5 hospitals of Guangdong Province.(3)Susceptibility tests used (K-B disk diffusion method) detection of the three gram-negative bacteria of nosocomial infection of 16 general kinds of antimicrobial drug resistance rate, and to select the resistance patterns of isolates of the same PFGE genotype comparison.(4)According to the literature and the NCBI sequence, select the target gene fragments were detected with high specificity to understand the strains carrying IntI, IntⅡ, IntⅢstatus.(5)Integron-positive strains of further primers to conserved sequences,variable region sequencing of PCR products, the results in the GenBank database for homology comparison to detect the variable region containing the resistance gene cassettes and carry Gene.(6)According to disinfectant resistant gene qacEΔ1 primers, applied PCR to detect qacEΔ1 carrying in Klebsiella pneumoniae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia. (7)Study on qacEΔ1 positive strains resistance to common disinfectants in hospitals, compared with qacEΔ1 negative strains and standard strain, by quantitative bactericidal test.(8) Statistical Method:We establish a database using Epidata3.0 and entry the date with double People. Data analysis use software named SPSS 13.0.Results(1) Bewteen January 2009 and June 2010,160 strains of Klebsiella pneumoniae,134 strains Stenotrophomonas maltophilia and 243 strains Pseudomonas aeruginosa were isolated from five three A-level comprehensive and specialized hospitals nosocomial infections in Guangdong Province. Guangzhou Chest Hospital, where the separation of Klebsiella pneumoniae and Pseudomonas aeruginosa up to, respectively, accounting for 32.5%(52/160) and 30.9%(75/243); Children's Hospital of Shenzhen City, the Stenotrophomonas maltophilia is the most, accounting for 35.8%(48/134). Shantou Central Hospital and the First Affiliated Hospital of Shantou University Medical College, separation of the three Gram-negative bacteria of nosocomial infection at the middle level, the First Affiliated Hospital of Guangdong College of Pharmacy the least number of isolates. The three age groups the incidence of Gram-negative bacteria infection trends were "U" type.<1 year of age group,66-75 years old group and 75 years of age group are infected with a high incidence of age. Help the elderly and infants and young children is the focus for prevention of nosocomial infection. By sections of the infection rate in this study to sort in descending order, were the first 6 ICU, neonatal wards, neurology, oncology, hematology. Thus, ICU, neonatal wards, respiratory medicine is the focus of hospital infection control departments. Klebsiella pneumoniae, Pseudomonas aeruginosa and Stenotrophomonas maltophilia nosocomial infection caused by living in the first four sites of infection were:respiratory tract, urinary tract, sepsis and surgery-related sites.(2)160 strains of Klebsiella pneumoniae,134 strains Stenotrophomonas maltophilia and 243 strains Pseudomonas aeruginosa DNA by restriction pulsed field gel electrophoresis in the image, the display 13 to 20 DNA fragments of different sizes, PFGE genome by Bionumerics V4.0 software for cluster analysis, similarity values between 25.1% and 100%. Klebsiella pneumoniae NO.3 and NO.11, NO.19 and NO.20,NO.22 and N0.23,N0.25 and N0.26,N0.54 and NO.64 strains isolated from Guangzhou Chest Hospital, NO.2 and NO.5, NO.4 and NO.8 strains isolated from Children's Hospital of Shenzhen City; Stenotrophomonas maltophilia NO.2 and NO.5 strains isolated from the First Affiliated Hospital of Guangdong College of Pharmacy, NO.l and NO.50,NO.7and NO.8, NO.28,NO.30 and NO.39 strains isolated from Children's Hospital of Shenzhen City, NO.50 and NO.61 strains isolated from Shantou Central Hospital; Pseudomonas aeruginosa NO.3and NO.8 strains isolated from the First Affiliated Hospital of Guangdong College of Pharmacy, NO.38and NO.39, NO.47and NO.57, NO.27and NO.62 strains isolated from Guangzhou Chest Hospital, NO.8and NO.19. NO.64and NO.65, NO.23,NO.24and NO.37, NO.2. NO.3and NO.9 strains isolated from Children's Hospital of Shenzhen City; NO.3and NO.4, NO.17and NO.18, NO.10and NO.13 strains isolated from Shantou Central Hospital completely homologous, occurs in the same room or same hospital ward; other strains showed a low homology or no homology.(3)Klebsiella pneumoniae antibiotics resistance rate to more than 50% are ampicillin, piperacillin, cephalexin, cefazolin, aztreonam, cotrimoxazole, ceftazidime and gentamicin; Stenotrophomonas maltophilia, in addition to co-trimoxazole, cefepime, ciprofloxacin, levofloxacin, and piperacillin/tazobactam, the antibiotics resistance rate of over 50%; the order of Pseudomonas aeruginosa resistance antibiotics more than 50% is co-trimoxazole, cefazolin, ampicillin, cefotaxime, cephalexin, and piperacillin. The resistance rate of strains isolated from different hospital showed statistically significant differences.(4) NO.3 and NO.11 strains isolated from Guangzhou Chest Hospital, showed exactly the same resistance spectrum, the other 4 pairs show no regular different,in 5 pairs of completely homologous Klebsiella pneumoniae strains, It shows that the same genotype can show different resistance phenotypes. Meanwhile, NO.37 and NO.51 strains show identical resistance spectrum, however, by clustering analysis, the similarity value is only 68.8%. Select the same resistance spectrum 7 Stenotrophomonas maltophilia, and 22 strains of Pseudomonas aeruginosa by PFGE pattern cluster analysis and found that there are such a similar situation.(5)30 strains of Klebsiella pneumoniae were detected in 11 strains (36.67%) (11/30) carried IntⅠ,17 strains (56.67%) (17/30) carried IntⅡ, were not detected in classⅢintegron; 30 Stenotrophomonas maltophilia were detected in 7 strains (23.33%) (7/30) carried IntI, was not found to carryⅡ,Ⅲintegron gene; 30 strains of Pseudomonas aeruginosa were detected in 23 (76.67%) (23/30) carried IntⅠ,7 strains carry (23.33%) (7/30) IntⅡ,1 strain (3.33%) (1/30) carry IntⅢgene.(6)Which are positive IntⅠand IntⅡKlebsiella pneumoniae strains for the First Affiliated Hospital of Guangdong College of Pharmacy's NO.2037 and NO.2997, and the Chest Hospital of Guangzhou, NO.21 strains. With 7 strains of Pseudomonas aeruginosa IntⅠand IntⅡwere positive, respectively, the First Affiliated Hospital of Guangdong College of Pharmacy NO.2669, Guangzhou Chest Hospital's NO.15 and NO.24, and Shenzhen Children's Hospital of NO.3,NO.9,NO.26,NO.27 strains. IntI and IntⅢwere positive for Pseudomonas aeruginosa, for the First Affiliated Hospital of Guangdong College of Pharmacy's NO.3159 strains.(7)The of length of variable region of IntⅠpositive Klebsiella pneumoniae is 1155bp, IntI positive Stenotrophomonas maltophilia is 4980bp, IntI positive Pseudomonas aeruginosa is 2420bp. The length of variable region of IntⅡpositive Klebsiella pneumoniae is 1795bp, IntⅡpositive Pseudomonas aeruginosa is 2870bp. The length of variable region of IntⅢpositive Pseudomonas aeruginosa is 2513bp.(8)The amplified products of the variable region sequencing and sequence analysis were obtained 6 integron resistance gene cassettes in combination, which IntⅠpositive Klebsiella pneumoniae integron sequenced by a combination of "unknown" forms of resistance gene cassettes, Whether it is new integron gene cassettes found in combination, to be landed comparison. IntⅡpositive Klebsiella pneumoniae sequence are "unknown+aadA4" resistance gene cassettes in combination, in which aadA (aminoglycoside antibiotics on the resistance) gene cassette is the most common gene cassette.Blast the two unknown fragment of the Internet than the latter, and Genbank storage no significant similarity of known sequence (nucleic acid similarity<85%), can be judged as the newly discovered gene, its function is to be further analysis. IntⅠpositive Stenotrophomonas maltophilia ClassⅠintegrons were sequenced by "aac-7+ Oxa-7+blap+aadB+ORF-E "gene cassette combinations of resistance, the strains of Stenotrophomonas maltophilia resistant Potency analysis revealed that the addition of piperacillin/tazobactam-sensitive, but to the aminoglycosides,βlactams, quinolones, carbapenems, sulfonamides such as 15 kinds of antibiotic resistance seriously. Randomly select a IntⅠpositive, a IntⅡpositive and a IntⅢpositive Pseudomonas aeruginosa variable region of the integration of positive amplification products and sequencing. The results show, classⅠintegron obtained by sequencing aac-3+aad2 form of combination;classⅡintegron resistance gene by sequencing by CmlA box; classⅢintegron resistance gene by bla-MAP box. One aad-3 and aad2 to aminoglycoside antibiotic resistance gene cassette, CmlA for Chloromycetin and Bla-MAP for the metallo-β-lactamase gene cassette, With three strains of Pseudomonas aeruginosa were resistant properties of the corresponding.(9)QacEΔ1 disinfectant resistant gene in 30 strains of Klebsiella pneumoniae were detected in 11 positive (36.67%) (11/30); in 30 strains of Stenotrophomonas maltophilia were detected in 4 positive (13.33%) (4/30); in 30 strains of Pseudomonas aeruginosa,26 strains were detected positive (86.67%) (26/30).(10)Compared with the standard strain of Pseudomonas aeruginosa, the effective iodine concentration of 200mg/L and 150mg/L, the role of the most time 7min, there are still 7.7% and 15.4% of qacEΔ1-positive and 6.7% and 10.0% qacEΔ1-negative strains of Pseudomonas aeruginosa survival respectively. Benzalkonium chloride concentration of 300mg/L and 250mg/L, the role of the most time 7min, there are 11.5% and 15.4% of qacEΔ1-positive and 6.7% and 20.0% of qacEΔ1-negative strains survival respectively. Chlorine concentration of 500mg/L and 375mg/L, the role of the most time 10min,19.2% and 38.5% still qacEΔ1-positive and 10.0% and 36.7% qacEΔ1-negative strains survive respectively.Conclusions (1)160 strains of Klebsiella pneumoniae,134 strains of Stenotrophomonas maltophilia and 243 strains of Pseudomonas aeruginosa hospital infection in Guangdong Provice,<1 year of age group,66-75 years old group and 75 years of group are infected with a high incidence of age. Distribution department, were the first 6 ICU, neonatal wards, neurology, oncology, hematology; sputum specimens is the main source. In the first 4 sites of infection were:respiratory tract, urinary tract, sepsis and surgery-related sites.(2)Molecular epidemiology analysis revealed that 5 hospitals in Guangdong Klebsiella pneumoniae, Stenotrophomonas maltophilia and Pseudomonas aeruginosa for the polyclonal composition, showed gene polymorphism, indicating that the hospital infection is disseminated during monitoring.(3)160 strains of Klebsiella pneumoniae,134 strains of Stenotrophomonas maltophilia and 243 strains of Pseudomonas aeruginosa are mostly multi-resistant strains. Comparison PFGE typing and drug resistance on the spectral can be seen that the same clonal type strains of antibiotic resistance patterns is vary, and different types of strains can be cloned with the same spectrum of antibiotic resistance, suggesting that strains of genotype between the antibiotic resistance spectrum is not necessarily related.(4)This study found that type I integron highest detection rate is in Pseudomonas aeruginosa,type II integron highest detection rate is in Klebsiella pneumoniae, Stenotrophomonas maltophilia was not found to carryⅡ,Ⅲintegron Gene. Of which 30 strains of Pseudomonas aeruginosa detected a strain (3.33%) carry IntⅢgene, for the first report.(5)The amplified products of the variable region sequencing and sequence analysis were obtained 6 integron resistance gene cassettes in combination, including aad-3 and aad2 to aminoglycoside antibiotic resistance gene cassette, CmlA for Chloromycetin and Bla-MAP for the metallo-β-lactamase gene cassette. Blast the two unknown fragment of the Internet than the latter, and Genbank storage no significant similarity of known sequence (nucleic acid similarity<85%), can be judged as the newly discovered gene, its function is to be further analysis.(6)disinfectant resistant gene qacEΔ1 in 30 strains of Klebsiella pneumoniae were detected 11 cases positive (36.67%); in 30 strains of Stenotrophomonas maltophilia were detected 4 positive (13.33%); in 30 strains of Pseudomonas aeruginosa were detected 26 positive (86.67%).(7)The disinfectant resistant gene qacEΔ1-positive Pseudomonas aeruginosa to povidone-iodine, benzalkonium chloride and chlorine disinfectant resistance is stronger than qacEΔ1-negative strains and the standard strain.