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Objective:To explore the serotype, antibiotic resistance and β-lactamase gene of Haemophilus influenzae strains isolated from hospitalized children, thus providing reference for clinical diagnosis and treatment. Methods:A total of 148 Haemophilus influenzae strains collected from January 2016 to December 2018 in hospitalized children of Children′s Hospital, Capital Institute of Pediatrics were retrospectively analyzed.The serotype and genotype of Haemophilus influenzae strains were examined by slide agglutination test and PCR, respectively.The sensitivity of isolates to Ampicillin and other antimicrobials was detected by the E-test and disk diffusion methods.The β-lactamase phenotype was tested by nitrocefin disk method.The carrying of β-lactamase gene TEM-1 and ROB-1 were detected by PCR.The drug resistance rate was compared by χ2 test. Results:All the 148 strains were nontypeable Haemophilus influenzae (NTHi), and capsular gene was not amplified.The rate of resistance to Ampicillin, Ampicillin/Sulbactam, Cefuroxime, and Azithromycin were 68.9%(102/148 strains), 40.5%(60/148 strains), 53.4%(79/148 strains) and 56.1%(83/148 strains), respectively.The Haemophilus influenzae isolates showed the highest resistance rate to Trimethoprim-sulfamethoxazole, which was up to 91.9%(136/148 strains). The sensitive rate of isolates to Ceftriaxone, Meropenem and Levofloxacin were all 100.0%(148/148 strains). The prevalence of β-lactamase was 64.8%(96/148 strains) in Haemophilus influenzae and the genotype was TEM-1.The drug resistance rates of β-lactamase positive strains to Ampicillin, Ampicillin/Sulbactam and Azithromycin were significantly higher than those of other strains( χ2=123.222, 27.973, 70.273, all P<0.01). Conclusions:The most prevalent serotype of Haemophilus influenzae is NTHi in children. Haemophilus influenzae carried TEM-1 gene had a high positive rate of β-lactamase production, which was the main mechanism of drug resistance to Ampicillin.Ceftriaxone and Meropenem were the most active agents against Haemophilus influenzae.
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Objective:To compare the in vitro susceptibility of fluconazole-resistant Candida albicans strains from superficial and deep infections to 8 antifungal drugs, and to compare drug resistance mutations in these strains. Methods:According to the Clinical and Laboratory Standards Institute (CLSI) protocol M27-A4, 26 deep infection-derived and 33 superficial infection-derived drug-resistant Candida albicans strains were tested for in vitro susceptibility to 8 antifungal drugs (fluconazole, voriconazole, itraconazole, posaconazole, amphotericin B, fluorocytosine, terbinafine, and micafungin) alone or in combination. DNA was extracted from all drug-resistant strains, and mutations in 3 drug resistance genes, including ERG3, ERG11 and FUR1, were detected by PCR. Normally distributed measurement data with homogeneous variance were compared between two groups by using two-independent-sample t test, non-normally distributed measurement data with non-homogeneous variance were compared using Mann-Whitney U test, and enumeration data were compared using chi-square test. Results:The minimum inhibitory concentrations (MICs) of fluconazole, itraconazole, voriconazole, posaconazole and fluorocytosine all significantly differed between the superficial infection group and deep infection group (all P < 0.05) , while there was no significant difference in the MIC of amphotericin B or micafungin between the two groups (both P > 0.05) . The MIC of terbinafine was >64 μg/ml in 96.6% of the above strains, so could not be compared between groups. As combination drug susceptibility testing revealed, the combination of terbinafine with azoles (fluconazole, voriconazole, itraconazole or posaconazole) showed synergistic inhibitory effects against 15 Candida albicans strains (7 strains from deep infections, 8 strains from superficial infections) , with fractional inhibitory concentration (FIC) indices being 0.033 to 0.187; no marked synergistic effect was observed in the combinations between fluorocytosine and azoles, between fluorocytosine and amphotericin B, or between amphotericin B and fluconazole, with the FIC indices being 0.56 to 1.125. The missense mutation V351A in the ERG3 gene was identified in all the 33 (100%) superficial infection-derived strains, as well as in 13 (50%) deep infection-derived strains, and the mutation A353T in the ERG3 gene was identified in 4 (15%) deep infection-derived strains; as for the ERG11 gene, missense mutations identified in the superficial infection-derived strains included I437V (32 strains, 97%) , Y132H (23 strains, 70%) , T123I (16 strains, 48%) , K128T (6 strains, 18%) , D116E (5 strains, 15%) , A114S (4 strains, 12%) , E266D (2 strains, 6%) , G448E (2 strains, 6%) , and G465S (2 strains, 6%) , while missense mutations identified in the deep infection-derived strains included I437V (23 strains, 88%) , E266D (13 strains, 50%) , E260G (5 strains, 19%) , and V488I (4 strains, 15%) ; the missense mutation R101C in the FUR1 gene was identified in 11 (33%) superficial infection-derived strains, but not identified in deep infection-derived strains. Conclusion:The drug susceptibility and drug resistance mutations differed to some extent between superficial infection- and deep infection-derived fluconazole-resistant Candida albicans strains.
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Objective:To investigate clinical and bronchoscopy characteristics of Mycoplasma pneumoniae pneumonia in children with 23S rRNA resistance gene positive in bronchoalveolar lavage fluid(BALF), and find clinical indicators that can identify Mycoplasma pneumoniae drug resistance early.Methods:The clinical data of 61 hospita-lized children diagnosed with Mycoplasma pneumoniae pneumonia as subjects from October 2017 to June 2018 in the Department of Pediatric Respiratory Medicine of Shengjing Hospital Affiliated to China Medical University were analyzed retrospectively.Bronchoscopy was performed on each subject and the BALF was taken and used to detect the 2063 site mutation of the 23S rRNA V region gene in BALF, and they were divided into drug-resistant gene positive group and drug-resistant gene negative group.The clinical manifestations, relevant laboratory data, imaging data, and bronchoscopy findings in different load groups were compared.Statistical methods such as t test, rank sum test, χ2 test, Fisher′ s exact probability method, and multivariate Logistic regression analysis were used to analyze the data. Results:Among the 61 children, 38 cases (62.30%) were in the drug resistance gene positive group, and 23 cases (37.70%) were in the drug resistance gene negative group.There were no significant differences in gender and age between the two groups (all P>0.05). The days of hospitalization and fever in the children with positive drug resistance genes were longer than those in the negative drug resistance gene groups, and they were more likely to have refractory mycoplasma pneumoniae pneumonia(RMPP) and extra pulmonary complications, with statistically significant differences ( P<0.05), but there were no significant differences in hypoxemia ( P>0.05). There were significant differences in white blood cell(WBC), C-reactive protein(CRP), procalcitonin(PCT), D-Dimer (D-D) and interleukin-6 (IL-6) between the two groups (all P<0.05). Except that the WBC level in the drug-resistant gene-positive group was lower than that in the drug-resistant gene-negative group, the rest of the test results indicated that the drug-resistant gene-positive group was higher than the drug-resistant gene-negative group.There were no significant differences in the concentrations of serum lactate dehydrogenlase(LDH)and IL-6 in BALF ( P>0.05). In this study, Logistic regression analysis was performed on several statistically significant laboratory indicators.It was found that WBC was more sensitive to identify drug resistance genes, and the optimal critical value was 8.55×10 9/L.The specificity of D-D in identifying drug resistance genes was higher, and the optimal cut-off value was 523 μg/L.In the drug resistance gene positive group, 35 cases (92.11%) showed extensive lung consolidation/atelectasis on imaging, and the drug resistance gene negative group was 13 cases (56.52%), with statistically significant differences ( P<0.05). The drug resistance gene-positive group mainly showed mucosal erosion, necrosis, phlegm plug/plastic phlegm plug and bronchitis stenosis, with a total of 19 cases (50.00%). In the drug resistance gene negative group, the main manifestations of mucosal longitudinal wrinkle, flocculent and viscous secretions were 14 cases (60.88%), with statistically significant differences ( P<0.05). Conclusions:The point mutation of 23S rRNA V region gene is closely related to the clinical characteristics of children with Mycoplasma pneumoniae pneumonia.Children with A2063G mutation are more prone to have RMPP and extrapulmonary complications, and their imaging manifestation and bronchoscopy are more severe.The levels of leukocytes and D-D in the blood have significance for the early identification of drug resistance.The systemic excessive immune inflammatory response caused by Mycoplasma pneumoniae pneumonia with drug-resistant gene positive needs to be valued.
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Objective To investigate the effect of silencing MFG-E8 gene on the sensitivity of SKOV3 cells to anticancer drugs and related mechanisms. Methods SKOV3 cells were transfected with MFG-E8 siRNA (Msi) and NC siRNA (Csi), respectively and the efficiency of transfection was confirmed by Western blot. The sensitivity of SKOV3 cells to cisplatin was observed by CCK-8 assay after transfection. The mRNA expression of ABCB1 and ABCC1 were detected by qRT-PCR. Effect of silencing MFG-E8 on the expression of ETM-related protein was detected by qRT-PCR and Western blot. Results MFG-E8 siRNA could effectively silence the expression of MFG-E8 protein. With the increasing drug concentration, the proliferation inhibition rate of each group also increased, and the cell proliferation inhibition rate of MFG-E8 siRNA group increased significantly (P < 0.01). Compared with NC siRNA group, downregulation of MFG-E8 expression led to decreased SKOV3 cell proliferation at 48h or 72h after 3 μg/ml cisplatin treat ment (P < 0.05). qRT-PCR results showed that the mRNA expression of ABCB1 and ABCC1 in Msi group were significantly lower than those in Csi group. qRT-PCR and Western blot results showed that silencing MFG-E8 gene down-regulated the mRNA and protein expression of N-Cadherin, Vimentin and Snail and up-regulated the expression of E-Cadherin. Conclusion Silencing the MFG-E8 gene can increase the sensitivity of SKOV3 cells against anti-tumor drugs and down-regulate the mRNA expression of ABCB1 and ABCC1, which may be related to the inhibition of EMT progression.
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Objective@#To know the pre-treatment drug resistance ( PDR ) status of newly reported human immunodeficiency virus type 1 ( HIV-1 ) infected individuals in Wenzhou, so as to provide guidance for antiretroviral therapy ( ART ). @*Methods@# Totally 232 plasma samples of newly reported HIV-1 infected individuals who had not received ART were collected in Wenzhou in 2019. Virus ( HIV-1 ) RNA was extracted, followed by reverse transcription PCR and nested PCR to amplify the pol region and sequence. Resistance mutations and resistance to non-nucleoside reverse transcriptase inhibitors ( NNRTIs ), nucleoside reverse transcriptase inhibitors ( NRTIs ) and protease inhibitors ( PIs ) was analyzed.@*Results@#The pol region sequences from 199 infected patients were obtained and the incidence of PDR was 8.04% ( 16/199 ). Eight genotypes were detected, including circulating recombinant forms ( CRFs ) CRF07_BC ( 47.24%, 94/199 ) and CRF01_AE ( 29.15%, 58/199 ) which were the dominant types. Two unique recombinant forms ( URFs ) were detected, namely URF( CRF01_AE/BC ) and URF( B/C ) . Thirty-one cases ( 15.58% 31/199 ) had drug-resistant mutations. For NNRTIs, NRTIs and PIs, 20 cases ( 64.52% ) , 2 cases ( 6.45% ) and 9 cases ( 29.03% ) with drug resistance mutations were detected, respectively. The resistance mutations to NNRTIs included K101E, K103N/R, V106I, E138K, V179D/E/T, Y181C, G190A and H221Y. Four cases each had two resistance mutations to NNRTIs. The resistance mutations to NRTIs were V75M and M184V. The resistance mutations to PIs were M46I, L33F and Q58E. For the newly released NNRTI drug Doravirine ( DOR ), two cases were found to have mutations of resistance. @*Conclusions@#The incidence of PDR among newly reported HIV-1 patients in Wenzhou is 8.04%, mainly caused by NNRTIs drug-resistant mutation. Resistance to the new drug DOR has emerged. The surveillance of drug resistance should continue to be strengthened.
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Objective@#To establish and evaluate a microfluidic chip platform for the rapid diagnosis of post-neurosurgical bacterial infection. @*Methods@#The pathogens isolated from patients with post-neurosurgical bacterial infection in Beijing Tiantan Hospital Affiliated to Capital Medical University during 2007 and 2016 and the epidemiological data from China drug resistance monitoring network CHINET were analyzed retrospectively. Based on the retrospective data and the molecular epidemiological information of drug-resistant bacteria reported in the literature, target pathogens and drug resistance gene parameters were selected. The microbial identification parameters from 10 different bacteria, including Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus epidermidis, Enterobacter cloacae, Staphylococcus aureus, Escherichia coli, Enterococcus faecium, Enterococcus faecalis, Stenotrophomonas maltophilia and Pseudomonas aeruginosa, and the parameters of 15 drug resistance genes, including mecA, vanA, vanB, aacC1, aadA1, bla CTX-M-1 , bla CTX-M-9 , bla GES-1 , bla OXA-23 , bla OXA-24 , bla OXA-58 , bla OXA-66 , bla KPC-2 , bla IMP-4 and bla VIM-2 , were selected for designing a microfluidic chip platform. Using MAIDI-TOF MS for bacterial identification, multiplex PCR for the detection of drug resistance genes, micro-broth dilution method for the detection of drug resistance phenotypes and ESBLs screening test as reference methods, 13 known bacteria were used to evaluate the preliminary performance of the established microfluidic chip platform, and 108 cerebrospinal fluid bacterial culture positive specimens were used to evaluate the clinical application value of the microfluidic chip platform. @*Results@#The identification rates of 13 known strains and the coincidence rate of drug resistance genes were 100%. The coincidence rate of identification results for 108 cerebrospinal fluid bacterial culture positive specimens between the microfluidic chip platform and the MALDI-TOF MS method was as high as 94.44%. The coincidence rates of drug resistance phenotype of carbapenems, oxacillin, vancomycin, ESBLs and genotype between the microfluidic chip platform and the micro-broth dilution method or ESBLs screening test were above 90%. @*Conclusion@#The established microfluidic chip platform is fast and accurate, and has application value in microbial identification and the prediction of drug resistance, which may be used as an important supplementary method in the diagnosis of post-neurosurgical bacterial infection.
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Objective@#To analyze the susceptibility and distribution of β-lactamase encoding and efflux pump genes as well as the inhibitory effect of efflux pump inhibitors on the carbapenem resistance of the carbapenem resistant Klebsiella pneumoniae without producing carbapenemase (CRKPPC). @*Methods@#One hundred and eight strains of carbapenem non-susceptible Klebsiella pneumoniae were collected from our hospital during 2012-2014. The strains producing carbapenemase were screened by Mastdiscs combi Carba plus disc system. For CRKPPC strains, the susceptibilities to antimicrobial agents were determined by micro-dilution broth methods. PCR and DNA sequencing technology were used to analyze the prevalence of β-lactamase encoding extended-spectrum β-lactamase (ESBL) and plasmid mediated AmpC genes as well as efflux genes including acrAB-tolC, kexD, kdeA, kpnEF and kpnGH. The inhibitory experiments were implemented by using carbonylcyanide-m-chlorophenylhydrazone (CCCP) and Reserpine to observe the role of efflux pumps on the carbapenem resistance. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed to analyze the expression of outer membrane proteins OmpK35 and OmpK36. @*Results@#Twenty-six strains out of the 108 carbapenem non-susceptible Klebsiella pneumoniae were identified to be CRKPPC strains which displayed quite high resistance to β-lactam and high resistance to amikacin, gentamicin, levofloxacin and ciprofloxacin. Very good sensitivities were observed to tigecycline, polymyxin, ceftazidime/avibatan and aztreonam/avibatan. The high prevalence of bla CTX-M and bla SHV were also displayed with the prevalent rates being 76.9% and 57.7%, respectively. The loss of outer membrane proteins OmpK35 and OmpK36 with varying degrees was observed, among which 57.7% strains lost ompK35 and 19.2% strains lost OmpK36. More than 80% strains contained efflux genes including acrAB-tolC, kexD, kdeA, kpnEF and kpnGH. The results of inhibitory experiments showed that CCCP displayed quite obviously inhibitory effects on the carbepenem resistance whereas no inhibitory effect was observed for Reserpine. @*Conclusion@#Tigecycline and polymyxin could be used as the basis of combined drug for CRKPPC. The prevalence of AmpC, ESBLs and loss of OmpK35 and OmpK36 with varying degrees among these strains were observed, but the over-expression of efflux pumps should be the main mechanism of the carbapenem resistance in the Klebsiella pneumoniae strains, and efflux inhibitors may have potential value in treating the infections caused by these bacteria.
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Objective To investigate the drug resistance and the distribution situation of the related drug resistant genes in Staphylococcus aureus (SA), and to provide a basis for the clinical rational use of antibiotics and the hospital control of infection. Methods A total of 135 strains of SA were collected from the Second Affiliated Hospital of Baotou Medical College during January to December 2017. BD Phoenix TM-100 automatic microorganism identification and drug sensitivity systems and K-B agar diffusion method were used to identify SA colony and analyze its drug susceptibility; the related drug resistant genes were detected by polymerase chain reaction (PCR). Results Among 135 strains of SA, 16 (11.9%) methicillin-resistant SA (MRSA) and 119 (88.1%) methicillin-sensitive SA (MSSA) were detected. In the 14 strains of MRSA, the resistance rates to ampicillin, penicillin and erythromycin were high (91.9%, 91.1% and 64.4%, respectively), and no vancomycin, teicoplanin and linezolid-resistant strains were found. Additionally, the resistance rates of MRSA to ciprofloxacin were significantly higher than that of MSSA [31.3% (5/16) vs. 5.0% (6/119), P < 0.05]. Among 135 strains of SA, the detection rates of mecA, aac(6')/aph(2"), erm(A), erm(B), erm(C), and tetM were 4.4% (6/135), 10.4% (14/135), 0.7% (1/135), 27.4% (37/135), 31.4% (46/135) and 0.7% (1/135), respectively. In MRSA, the detection rates of mecA [37.5% (6/16) vs. 0 (0/119)], aac(6')/aph(2") [31.3% (5/16) vs. 7.6% (9/119)], and ermB [31.3% (5/16) vs. 26.9% (32/119)] were significantly higher than those in MSSA. It is noteworthy that the detection rate of mecA in MRSA was only 37.5% (6/16). Conclusions The MRSA detection rate of our hospital was below the national average level. The detection rates of resistance genes mecA, aac(6')/aph(2") and ermB were higher, which may be an important cause of drug resistance. Moreover, the detection of MRSA by mecA alone may lead to missed diagnosis, that should be paid attention to.
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Objective To analyze the antimicrobial resistance, distribution of resistance genes and staphylococcal cassette chromosome mec ( SCCmec) in 99 strains of mecA gene-positive Staphylococcus epi-dermidis strains isolated from early pregnancy cervical swabs and external environment in Beijing Chaoyang District from 2015 to 2016. Methods Kirby-Bauer disk diffusion method was performed to detect the sus-ceptibility of the 99 Staphylococcus epidermidis strains to cefoxitin. Microbroth dilution method was used to test their susceptibility to vancomycin, daptomycin, penicillin, erythromycin, compound sulfamethoxazole, tetracycline, ciprofloxacin, clindamycin, gentamicin and chloramphenicol. PCR was used to detect drug re-sistance genes of ermA, ermB, ermC, msrA, norA1, norA2, sul1, sul2, sul3, aac(6')/aph(2″), ant(4', 4″), ant(6) and tetM and to analyze the SCCmec types ofⅠ, Ⅱ, Ⅲ, Ⅳa, Ⅳb, Ⅳc, Ⅳd and Ⅴ. The results were compared with those of capillary electrophoresis. SPSS was used for data analysis. Results All of the 99 mecA-positive Staphylococcus epidermidis strains were sensitive to vancomycin and 93. 94％ of them were sensitive to datomycin. The resistance rates to penicillin, erythromycin, cefoxitin, compound sulfame-thoxazole, tetracycline, ciprofloxacin, clindamycin, gentamicin and chloramphenicol were 97. 98％, 85. 86％, 79. 80％, 52. 54％, 27. 27％, 43. 43％, 36. 36％, 23. 23％ and 11. 11％. The strains that car-ried the genes of norA1, norA2, ermA, ermB, ermC, msrA, sul1, sul2, sul3, aac(6')/aph(2″), ant(4', 4″), ant(6) and tetM accounted for 100％, 93. 94％, 0. 00％, 3. 03％, 17. 17％, 57. 58％, 50. 51％, 12. 12％, 4. 04％, 30. 30％, 8. 08％, 4. 04％ and 25. 26％, respectively. Among the 99 strains, 5. 05％, 0％, 43. 43％, 10. 10％, 0. 00％, 3. 03％, 3. 03％ and 19. 19％ belonged to SCCmecⅠ, Ⅱ, Ⅲ, Ⅳa,Ⅳb,Ⅳc,Ⅳd andⅤ, respectively, and 4. 04％ (4/99) were positive to two SCCmec types. The types of 12. 12％ (12/99) of the strains were unidentified. Conclusions All of the 99 strains of mecA-positive Staphylococcus epidermidis were sensitive to vancomycin. Among them, the strains carrying multidrug resist-ance genes accounted for 89. 90％. The main mechanisms of resistance to macrolides, sulfonamides and ami-noglycosides in local strains were related to the resistance genes of msrA, sul1 and aac ( 6')/aph ( 2″) . SCCmec Ⅲ was the prevalent type. There were 88. 37％ of SCCmec Ⅲ type strains and 75％ of unknown type strains carrying multiple resistance genes. Apart from that, the isolated strains of other SCCmecⅢtypes all carried multiple resistance genes.
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Objective@#To monitor the antimicrobial resistance and drug-resistance genes of Yersinia enterocolitis, Y. intermedia and Y. frederiksenii recovered from retailed fresh poultry of 4 provinces of China.@*Methods@#The susceptibility of 25 isolated Yersinia spp. to 14 classes and 25 kinds of antibiotics was determined by broth microdilution method according to CLSI (Clinical and Laboratory Standards Institute). The antibiotic resistance genes were predicted with antibiotic resistance genes database (ARDB) using whole genome sequences of Yersinia spp.@*Results@#In all 22 Y. enterocolitis tested, 63.7% (14 isolates), 22.8% (5 isolates), 4.6% and 4.6% of 1 isolates exhibited the resistance to cefoxitin, ampicillin-sulbactam, nitrofurantoin and trimethoprim-sulfamethoxazole, respectively. All the 25 isolates were multi-drug resistant to more than 3 antibiotics, while 64.0% of isolates were resistant to more than 4 antibiotics. A few Y. enterocolitis isolates of this study were intermediate to ceftriaxone and ciprofloxacin. Most Yersinia spp. isolates contained antibiotic resistance genes mdtG, ksgA, bacA, blaA, rosAB and acrB, and 5 isolates recovered from fresh chicken also contained dfrA1, catB2 and ant3ia.@*Conclusion@#The multi-drug resistant Yersinia spp. isolated from retailed fresh poultry is very serious in the 4 provinces of China, and their contained many kinds of drug-resistance genes.
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Objective To investigate drug resistance genes and epidemic characteristics of β-lactamase carried by carbapenem-resistant Acinetobacter baumannii (CRAB) in the respiratory intensive care unit(RICU) in a hospital.Methods Clinically isolated CRAB from RICU patients in October-December 2015 were collected.Five drug resistance genes (KPC-2,IMP,VIM,NDM-1,OXA-23) were specifically amplified by polymerase chain reaction (PCR),amplified products were performed agarose gel electrophoresis and sequencing analysis,the homology was analyzed with pulsed-field gel electrophoresis (PFGE).Results A total of 22 CRAB strains were isolated in October-December 2015,19 (86.36％) of which were isolated from sputum.The resistance rate of 22 CRAB strains to compound sulfamethoxazole was 59.09 ％,resistance rate to minocycline was 9.09 ％,all were sensitive to polymyxin B,resistance rates to other antimicrobial agents were more than 80％.Three kinds of resistance genes KPC-2,IMP and NDM-1 were not found by PCR amplification,positive rates of VIM and OXA-23 were both 100％.PFGE homology analysis revealed that 22 strains were divided into 13 different types,each type contained 1-5 strains,9 types(69.23％) contained only 1 strain respectively,the other 4 types (30.77％) contained 2-5 strains.A5,A7,and A8;A9,A11,A14,A19 and A22;A4,A10 and A12;A16 and A18 were of the same type respectively.Conclusion The main types of β-lactamase-resistant genes of CRAB in RICU are VIM and OXA-23.Homology analysis shows a small parts are of the same clone strains,which reveals epidemic of a small scale.
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Objective To establishment a method for detection of multiple drug resistance gene of Yersina pestis using polymerase chain reaction(PCR), to provide a guidance for treatment of plague. Methods According to National Center for Biotechnology Information (NCBI) released sequences of aminoglycoside resistant genes of streptomycin resistant,strB,strA,beta lactam antibiotics resistant genes tem,shv,and ctx-m,sulfamilamide resistant genes sul1, sul2, and sul3, a pair of primers of each gene was designed. DNAs of 282 strains isolated from plague natural foci in Qinghai Province were amplified by PCR using every pair of primers. The products were separated using gel electrophoresis, and the results were visualized through a gel imaging system. The susceptibility of 282 Yersina pestis to streptomycin, sulfamethoxazole and ceftriaxone was tested by drug sensitivity test. Results The PCR amplification results of all samples were negative,and strains with streptomycin,sulfamilamide and beta lactam antimicrobial drug resistance genes were not found. Drug sensitivity test showed that 282 strains were highly sensitive to streptomycin,sulfamethoxazole and ceftriaxone sodium.The diameter of bacteriostasis ring>19,17,21 mm, respectively. Conclusions It is a feasible method to use PCR technology to detect the multiple drug resistance genes of Yersinia pestis. Using this method to systematically monitor the resistance gene of Yersinia pestis is an efficient, economical and practical experimental method, which can provide guidance for the treatment of plague disease.
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Objectives To investigate the homology and drug resistance gene of Group B Streptococcus ( GBS) Resistance induced by Clindamycin and provide basic data for clinical prevention and treatment of GBS Resistance infection induced by Clindamycin .Methods 921 strains of GBS were isolated at Obstetrics&Gynecology Hospital of Fudan University from January , 2014 to December , 2015.VITEK2-compact automatic bacterial susceptibility instrument was used to test their sensitivity to 7 antibacterial drugs.63 positive strains were chosen through D-inhibition zone trial which were drug resistant to Erythromycin and susceptible or intermediary to Clindamycin .The strain′s sequence type was identified by the method of multilocus sequence typing ( MLST typing ) .The drug resistance genes mefA & ermB to Erythromycin were detected by using PCR method .The analysis was carried out to reveal the relevance to drug resistance , multilocus sequence typing and drug resistance gene .Results Among 921 strains of GBS , the drug resistance rate was respectively 53.4％ ( 492/921 strains ) to Erythromycin , 50.2％ ( 462/921 strains) to Clindamycin, 34.7％ ( 320/921 strains ) to Levofloxacin and 7.5％ ( 69/921 strains ) to Nitrofurantoin.The drug resistance rate of Levofloxacin for 63 GBS strains was 27.0％(17/63 strains) and no drug resistant strain was found to Penicillin , Vancomycin & Nitrofurantoin.12 different ST types were involved in total, including a new ST type:ST1072.The most common ones were ST12 (30.1％) (20/63 strains) &ST19 (25.4％) (16/63 strains).The drug resistance rate of Levofloxacin with ST 19 (75.0％) (12/16 strains) was much higher than that of other ST types .The relevance ratio of mefA and ermB among 63 GBS strains was respectively 27.0％(17/63 strains) and 41.3％(26/63 strains).Conclusions The genetic diversity existed in Group B Streptococcus resistance induced by Clindamycin detected in this study . There was significant difference on drug resistance and relevant drug resistant genes among different ST types.
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Objective To analyze the aminoglycoside ( AG ) antibiotics resistance rate of carbapenem-resistant Klebsiella pneumoniae ( CRKP ) and its molecular mechanisms.Methods One hundred and four strains of CRKP isolated from 4 hospitals in Zhejiang Province from January 2013 to June 2014 were collected, including 56 strains from Sir Run Run Shaw Hospital , Zhejiang University School of Medicine ( S hospital ), 22 from the First Affiliated Hospital, Zhejiang University School of Medicine ( Z hospital), 13 from Yiwu TCM Hospitals (Y Hospital) and 13 from Fuyang First People's Hospital (F Hospital).VITEK 2 Compact method and K-B disk method were used to detect the susceptibility of commonly used antibiotics including three kinds of AGs (kanamycin, gentamycin and amikacin ).PCR and sequencing techniques were used to screen the aminoglycoside resistance -related 16S rRNA methylation genes (rmtA, rmtB and armA) and the aminoglycoside modified enzyme resistance gene [aac(6′)Ⅰb].The relationship between drug resistance and carrier status of drug resistance genes was analyzed .Homologous analysis of rmtB-positive strains was performed using PFGE to examine the epidemic spread of strains in each hospital.Results All 104 CRKP strains were multi-drug resistant and had high resistance to cephalosporins, fluoroquinolones ( ciprofloxacin, levofloxacin ) and nitrofurantoin.The resistance rates to gentamicin, kanamycin and amikacin were 73.1%(76/104), 64.4%(67/104) and 56.7%(59/104), respectively.The carrying rates of aminoglycoside-resistance genes were: rmtB 56.7%( 59/104 ), aac (6′)Ⅰb 17.3%(18/104), armA 1.9%(2/104); while no rmtA was detected.Thirty-seven strains did not carry the screened genes.Amikacin-resistant strains were resistant to both kanamycin and gentamicin, and both were rmtB-positive strains.The PFGE classification results showed that 104 strains were divided into 11 clonal populations, and there were scattered non-population clones in each hospital. There were seven major clonal populations (Ⅰ-Ⅶ) carrying rmtB genes, of which typeⅠ, typeⅢand typeⅤwere prevalent in S hospital ; typeⅡ, typeⅥand TypeⅦwere popular in Z hospital ; the distribution of strains in Y hospital was scattered ; F hospital had one independent clone type Ⅳ(3 strains).Conclusion AGs still have certain sensitivity to CRKP strains.The main mechanism of strain resistance to AGs is the rmtB gene-mediated 16S rRNA methylase.
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OBJECTIVE:To provide reference for rational drug use in clinic and nosocomial infection control. METHODS:Acinetobacter baumannii(AB)were collected from our hospital during Jan. 2014-Jun. 2017. Drug sensitivity tests were conducted by using K-B method and MIC method. Drug-resistance genes of multidrug-resistant Acinetobacter baumannii(MDR-AB)were amplified by PCR,and compared with GenBank database by using Blast comparison. RESULTS:A total of 1 758 strains of AB were detected,and mainly came from sputum and throat swab(65.24%),followed by urine(18.49%). These infected patients were mainly distributed in the departments of ICU(38.51%)and respiratory medicine(24.00%),respectively. Drug resistance of clinical isolated AB to most commonly used antibiotics were more than 40%,such as compound sulfamethoxazole,piperacillin sodium and tazobactam sodium,gentamicin,cefepime,levofloxacin,minocycline,imipenem,etc.;it had increased year after year. Drug resistance to colistin was lower than 5% and decreased year by year.A total of 673 strains of MDR-AB were detected, and detection rates were 22.77%,29.82%,52.09%,54.33%,respectively.Among 110 strains of MDR-AB,detection rates of TEM, AmpC,IMP,VIM,OXA-23,OXA-24,OXA-51,aac(6′)-Ⅰ,aac(3)-Ⅰ,ant(3″)-Ⅰ,anmA,gyrA,parC gene were 97.27%, 91.82%,49.09%,12.73%、90.91%,12.73%,98.18%,34.55%,60.91%,89.09%,87.27%,77.27%,82.73%,respectively. Results of Blast comparison showed that point mutation occurred in 83rd and 121st base of gyrA gene,144th base of parC gene. CONCLUSIONS:AB mainly come from sputum and throat swab specimens in our hospital,and infected patients are mainly distributed in the departments of ICU and respiratory medicine. Drug resistance is serious,and the detection rate of MDR-AB is increased year by year. Main genes of multidrug-resistant strains mainly include TEM,AmpC,OXA-23,OXA-51,ant(3″)-Ⅰ, anmA,etc.,and mutation of gyrA and parC gene are found. It is necessary to strengthen the management of classification use of antibiotics and strengthen the monitoring of AB drug resistance. According to the results of drug sensitivity test,antibiotics are selected rationally to prevent or delay planting and cross transmission of AB-resistant strain.
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Objective To investigate the class I integrons and their gene cassettes of imipenem-resistant Pseudomonas aeruginosa (IRPA) , and to analyze the correlation between integrons and drug resistance. Methods PCR was used to determine the presence of integrase genes and class I integrons. The variable regions were detected by sequencing. Resistance genes of integron gene cassettes including metal-β-lactamases, aminoglycoside modifying enzymes (AMEs), 16SrRNA methylating enzyme and the OprD2 genes were detected by PCR. The VITEK-2 automated system was used to determine the antibiotic susceptibility of integron-positive IRPA strains. Results The positive rates of integrase genes and class I integrons were 23.3%(20/86)and 8.14%(7/86) , and five kinds of gene cassettes were detected in 86 IRPA strains. The class I integrons-positive bacterial strains exhibited different resistant patterns to 12 antibiotics with large number of resistance genes. Conclusion The class I integrons and their gene cassettes are associated with multiple drug resistance of IRPA.
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Objective:To assess the prevalence of HIV primary drug resistance and drug resistance gene mutations among men who have sex with men (MSM).Methods:We searched eight electronic databases (CNKI,VIP,CBM,Wanfang Database,PubMed,Web of Knowledge,Springer,Medline) for the studies of HIV drug resistance relevant to MSM.Drug resistance and drug resistance mutations data were pooled and analyzed according to statistical test of homogeneity.Subgroups were further divided according to sample size,location,race,quality rating score,sampling time.Results:Forty-three studies were included in this Meta-analysis.The pooled rate of total to protease inhibitor (PI),nucleoside reverse transcriptase inhibitor(NRTI) or non-nucleoside reverse transcriptase inhibitor (NNRTI) were 10.21％ (95％ CI 8.65％ to12.03％),2.98％ (95％ CI 2.25％ to 3.93％),4.05％ (95％ CI 3.14％ to 5.21％),4.42％ (95％ CI 3.31％ to 5.88％),respectively.The pooled rates of PI major mutation,PI secondary mutations,NRTI mutations and NNRTI mutations were 0.55％ (95％ CI 0.38％ to 0.80％),1.31％ (95％ CI 0.98％ to 1.75％),0.85％ (95％ CI 0.51％ to 1.40％),1.19％ (95％ CI 0.70％ to 2.01％),0.79％ (95％ CI 0.55％ to 1.13％),1.73％ (95％ CI 1.21％ to 2.46％),0.86％ (95％ CI 0.61％ to 1.21％),2.24％ (95％ CI 1.77％ to 2.83％),respectively.Sample size,region,and race were heterogeneous sources;the rate of resistance mutations and gene mutation rate were different in different subgroups.Conclusion:The prevalence of primary drug resistance among MSM was high in Americas and Europe,and it was gradually increased in Asia.We should pay attention to the high incidence of PI secondary mutations.
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Objective To establish the in vitro biofilm model of Laribacter hongkongensis(LH),to analyze the type Ⅰ integron related genes carried by LH and to investigate the mechanism of LH resistance to quinolones.Methods The biofilm forming abilities of LH clinical isolates were determined by Giemsa staining qualitative method and by crystal violet staining semi-quantitative method.The sensitivity of LH to norfloxacin,ofloxacin,levofloxacin,ciprofloxacin and lomefloxacin in both planktonic and biofilm conditions were dectermined by broth microdilution susceptibility tests.Type I integron related genes carried in 18 LH strains resistant to quinolone were detected by PCR amplification method.Results The detection results by Giemsa staining demonstrated that 36 strains in 55 LH clinical isolates formed visible biofilm,and the biofilm formation rate was 65.4%(36/55).In the biofilm forming ability detected by crystal violet staining semi-quantitative method,OD560≤0.15 was in 8 strains of LH,0.150.20 in 7 strains respectively.The levels of minimal biofilm inhibitory concentration in norfloxacin,ofloxacin,levofloxacin,ciprofloxacin and lomefloxacin to LH were respectively higher than the minimal inhibitory concentration(MIC) in the corresponding floating bacteria(P<0.05).Among 55 strains of LH,18 strains were resistant to quinolones,the resistance rate was 32.7%,and the type I integron in 18 strains of LH carried the drug resistant genes,these drug resistant genes played the drug resistance role in corresponding bacterial strains.Conclusion Drug resistance gene formation and widespread of LH may be associated with type I integron.
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Objective To understand the mutations of macrolide resistance gene locus (23S rRNA) of Mycoplasma pneumoniae (MP) and its correlation with clinical features .Methods A total of 354 respiratory tract samples were collected from children pa-tients with pneumonia .MP and its mutations in 23S rRNA gene locus were detected by real-time PCR .The children cases of MP positive were divided into the mutation group and non-mutation group .Then the clinical data were compared between the two groups .Results Among 354 respiratory tract samples ,166 cases(46 .9% ) were MP positive ,moreover the mutation of 23S rRNA gene locus existed in 135 MP positive samples with the positive detection rate of 81 .3% ,while no 23S rRNA gene locus mutations were detected in 31 samples .Analyzing the clinical data of the mutation group and non-mutation group found that there was no sta-tistical difference in the aspects of age and gender between the two groups .The occurrence rates of severe pneumonia and extrapul-monary complications in the mutation group were higher than those in the non-mutation group (P<0 .05) ,moreover the average hospitalization time and fever duration in the mutation group were longer than those in the non-mutation group (P<0 .05) .Conclu-sion 23S rRNA gene locus mutation has higher detection rate ,prompting that MP shows high resistant rate to macrolides ,which could provide a certain basis for treatment of M P infections .
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Objective To analyze the distribution of common drug-resistance genotypes of multi-drug resistant bacteria (MDRB) in second class and below hospitals in 3 big areas of Chongqing City for perfecting the bacterial drug resistance surveillance network in local area.Methods In 7206 detected strains of MDRB, the re-cultured pure colonies of top five bacteria in the bacterial strains numer were taken and performed the common drug resistant genotyping detection and comparative analysis by PCR technique and sequencing.Results Acinetobacter spp.was dominated by the genotypes carrying TEM,SUL and GyrA genes,Klebsiella pneumoniae was dominated by the genotypes carrying SHV,GyrA genes,Escherichia coli was dominated by the genotypes carrying TEM,CTX-M,SUL,GyrA,aac (3) Ⅱ genes,Pseudomonas aeruginosa was dominated by genotypes carrying SUL,GyrA genes,Staphylococcus was dominated by genotypes carrying GyrA,aac (6 ′)-aph ′′ genes;among the five strains of MDRB,the majority were the strains with multiple expression of two kinds or four kinds of common drug-resistance genes,in which the detection rate of Escherichia coli multiple expression was highest,reaching 92.74%,the detection rate of Staphylococcus multiple expression was lower.The detection rates of common drug resistant genotypes carried by MDRB had statistical difference among various areas and various years (P<0.05);in the comparison with the gene sequences of corresponding bacteria in NCBI Blastn database,the sequencing results of 7 common drug resistant genotypes carried by 5 kinds of MDRB were basically consistent.Conclusion The common drug-resistant genotypes carried by MDRB detected in the second class and below hospitals of Chongqing City and their distribution are basically consistent with the monitoring levels in the local tertiary hospitals and whole nation.Therefore the antibacterial surveillance of infection pathogenic bacteria should be strengthened in these hospitals,and medication should be rationally used so as to delay the development of pathogenic bacterial drug resistance in local area.