| Bacterial resistance to antibiotics has become a global concern. Infection of resistant bacteria has posed a new challenge to anti-infective therapy in new century, and a major threat of human health and life currently. Enterobacteriaceae bacteria is widely distributed, and has a wide host range from human being to animals and plants,with a status of parasitic, symbiotic, epiphytic and saprophytic. They can survive in soil and water, closely related to human. Among health-care associated infection, Enterobacteriaceae, including Escherichia coli, Klebsiella pneumonia, is the most common pathogen, and often characterized as multi-drug resistant bacteria. Carbapenem was the most effective antimicrobial agent for clinical treatment of multiple drug-resistant strains producing ESBL/AmpC enzymes. However, with the widespread and unreasonable use of these drugs, carbapenem-resistant organisms had emerged clinically. International reports about mechanism of resistance to carbapenems focused on three areas:?producing various carbapenemases, such as blaIMP, blaVIM, KPC, et al.?Overexpression of ESBL and/or AmpC enzyme combining with outer membrane porin loss.?Over-expression of efflux pump proteins to alter the membrane permeability;?arget alteration. In these mechanisms, the most prominent is enzyme production. Luo Jun et al found that from June 2003 to May 2004 in Huashan Hospital, among imipenem-resistant gram-negative bacilli, carbapenemase producing mechanism was mainly responsible for imipenem and meropenem resistance in Acinetobacter and Citrobacter. Following Shen and others analyzed the MIC of 199 gram-negative bacilli by agar dilution method, results showed the resistance rate of carbapenem-resistant Gram-negative bacilli against 12 antibiotics were higher than carbapenem-sensitive gram-negative bacilli, and CRE produced a variety of carbapenemases, such as KPC, IMP, VTM and OXA, et al., and may cause a clonal epidemic spread in Citrobacter, Acinetobacter baumannii and pneumonia speces. In Brazil, Enterobacteriaceae carbapenem resistance become a major problem, especially strains producing KPC enzymes have been reported in many areas. "Lancet" reported super bacteria producing NDM-1 enzyme, most saw in Escherichia coli and Klebsiella pneumoniae, and had spread to countries outside of India.These enzymes have a broad hydrolyzing spectrum of substrates, and become more frequency each year. During 2005-2008 in Huashan Hospital, the resistance rate of Klebsiella pneumoniae to imipenem, meropenem and ertapenem and other antimicrobial drugs was below 2%, however, in 2009, the rate rapidly increased to nearly 13%, and moreover, in 2010, the rate rose to 26.3%. For Citrobacter, in 2005 the carbapenem antibiotic resistance rate is near 10%, in 2006, it quickly rose to 45%, and kept at 35%during 2007-2009. Further study showed that KPC-type carbapenemases producing was mainly responsible for carbapenem resistance among Klebsiella pneumoniae in Huashan Hospital. As KPC gene was often carried by plasmid which often carried other resistance genes simultaneously, isolates producing KPC enzyme always are endowed with high level resistance and disseminated widely. Emerging and outbreak of such strains had occurred around the world. Many Hospitals in China, including Huashan Hospital, exhibited an increasing detection rate of CRE, and this leads to an embarrassing situation that no drugs available for patients.Based on the above situation, in the present study, we collected 82 unique isolates clinically from 1561 Enterobacteriaceae in Huashan Hospital during April 2009 to February 2010, and all were identified as carbapenem-resistant Enterobacteriaceae (CRE). Our aim was to clearly investigate the resistance mechanisms, distribution and dissemination mechanisms of these bacteria in our hospital, to provide reasonable selection and usage of antibiotics, reducing waste of large number of expensive agents. It is very important for in timely persistent infection control and the outbreak of carbapenem-resistance isolates, and reducing the incidence and mortality of health-care associated infection. The study included following four parts of contents.Part One:Antimicrobial susceptibility testing and homology analysis of carbapenem-resistant EnterobacteriaceaeTo understand the prevalence of CRE strains in Huashan Hospital, we collected 82 strains of CRE bacteria from 1561 Enterobacteriaceae, which were isolated during April 2009 to February 2010 from unique patients. Strains were analyzed by disk diffusion according to the 2010 updated version of the CLSI (Clinical and Laboratory Standard Institute) file, results showed 113 of 604 isolates Klebsiella pneumonia were CRE strains, which were the most common pathogen, followed by Escherichia coli, in which were found 12 of 581 isolates were CRE strains. Among 1561 isolates, Proteus mirabilis and Enterobacter cloacae each were about 5%, and 18.1% of Enterobacter cloacae isolates were CRE. The proportions of Serratia marcescens, Providencia stuartii, Morganella morganii, Klebsiella oxytoca, Proteus vulgaris, Enterobacter aerogenes and Providencia rettgeri were between 1-4%. Proportions of the remaining strains were below 1%. Isolates were mainly detected from spum and urine, accouting for 46.5%(725/1561) and 34.6%(540/1561) respectively. Male patients were 826, and female for 673, other 25 were loss of sex imformation. Patients were mostly between 40-80, accouting for 59.4%(927/1561).A total of 82 CRE strains were remained and used in the study, after cultured and recovered according to 2009 CLSI.82 CRE includes 68 strains of Klebsiella pneumonia,3 Escherichia coli,3 Enterobacter cloacae,4 Citrobacter freundii, one Enterobacter aerogenes, one Providencia stuartii, one Citrobacter malonate and one Klebsiella oxytoca. Minimum inhibit concentration was detected using agar dilution method for 16 drugs including three cabapenems, fluoroquinolones and cephalosporins against CRE strains, the results were interpreted according to the 2010 updated version CLSI criteria, except for colistin and tigecycline, which were interpreted by British Society for Antimicrobial Chemotherapy criteria, version9.1,March 2010.Antimicrobial susceptibility testing results showed that Resistance rates of 68 carbapenem-resistant Klebsiella pneumonia to ceftazidime, cefotaxime, cefepime, aztreonam and ertapenem were 100%, resistance rate of the two lactamase inhibitors (cefoperazone/sulbactam, piperacillin/tazobactam) were about 95%, of imipenem and meropenem were about 90%, of amikacin and ciprofloxacin were 85.3% and 97.1%. 70% of the 68 carbapenem-resistant Klebsiella pneumonia isolates were sensitive to fosfomycin and doxycycline. The resistance rates of colistin, minocycline and tigecycline were relatively lower, which were 2.9%,7.4% and 13.2%. The remaining 14 CRE were all resistant to ciprofloxacin and ertapenem. The resistance rates of two lactamase inhibitors were 76.9% and 61.5%. Nearly 70% of the 14 CRE isolates were sensitive to fosfomycin, and nearly 60% of 14 CRE were resistant to doxycycline. The resistance rates of colistin, minocycline and tigecycline were 15.4%,38.5% and 23.1%. Among all 16 antibiotics, clistin and tigecycline have the highest antibacterial activity with MIC90 values of 2?g/mL and 4?g/mL. Bacteria showed a high level of resistance to cephalosporin and carbapenems. The MIC50/MIC90 values of carbapenem antibiotics such as imipenem, meropenem, ertapenem were 32/64?g/mL, 64/128?g/mL and 128/256?g/mL.68 isolates showed resistance to all three carbapenems, of which 60 isolates belonged to Klebsiella pneumonia. MIC90 value of cephalosporin antibiotics were?256?g/mL. Our study showed that all 82 CRE strains were multi-drug resistant, and 54 were pan-resistant.Our data showed that before and after adding enzyme inhibitors, which were clavulanic acid and EDTA, into carbapenems, bacterial showed no grater bactericidal activity to 68 carbapenem-resistant Klebsiella pneumonia strains, however combined with 3-Aminobenzeneboronic acid, carbapenems has increased its bactericidal activity of bacteria, with MIC50/MIC90 of 1/l?g/mL,4/4?g/mL and 8/8?g/mL, compared with single drug, the MIC50/MIC90 value decreased as low as 1/32 to 1/64 of MICs of single drugs. It was also obvious in other 14 CRE. The vitro activity of carbapenem combined with the efflux pump inhibitor MC207110 were not effected on 68 CRE strains of Klebsiella pneumoniae, while on the other 14 CRE strains, the MIC50 values showed increases.Sixty-eight carbapenem-resistant Klebsiella pneumonia isolates could be classified into eighteen genotypes by PFGE using Quantity One image analysis software. There were three main types, namely J (20.6%,14/68), N(17.6%,12/68) and R(17.6%,12/68). Six isolates for Q type, five for I type, four for D type, two for Q H and P-type. Others like A, B, C, E, F, K, L, M and O type was one isolate respectively. The primary strains belonged to ST-11, accounting for 83.8%(57/68), ST-350 for 2 isolates, and ST-16, ST-22, ST-23, ST-27, ST-107, ST-148, ST-477, ST-494, ST-544 for one, respectively. J,N and R were all ST-11.This set data suggested:?carbapenem-resistant strains are mostly pan-resistant strains(65.9%,54/82).?colistin and tigecycline have high vitro activity for CRE strains.?Carbapenems combined with 3-Aminobenzeneboronic acid showed high vitro activity for CRE strains. Tthe MICs of carbapenems combined with efflux pump inhibitor to CRE did not decrease.?there are 3 major clonal bacterial strains, namely J-type (14 strains,20.6%), N-type (12 strains,17.6%) and R-type (12 strains, 17.6%). MLST showed ST-11 accounting for 83.8%(57/68). J,N and R all were ST-11. Part Two:Genotype analysis of CRE strainsModified Hodge test (MHT) and boric acid suppression test are used for carbapenemase phenotype screening. There were 67 isolates positively detected by MHT. Compared with PCR method, the sensitivity and specificity of carbapenemases detection of 82 CRE was 89.9%(62/69) and 61.5%(8/13); the false positive rate and the false negative rate were 7.2%(5/69) and 53.8%(7/13).3-amino-phenylboronic acid inhibition test showed a sensitivity and specificity of 92.7%(64/69) and 53.8% (7/13); the false positive rate and the false negative rate were 8.8%(6/69) and 38.5%(5/13). When detecting carbapenemase in Enterobacteriaceae bacteria, compared to PCR method, modified Hodge test and 3-amino phenyl boronic acid methods showed no significant difference in detection rate (P>0.05).82 CRE did not produce metal enzyme, as EDTA synergy test did not show any positive result.To understand the carbapenemase genotype of CRE strains, PCR method was used as goldstandard to detect carbapenemase genes. Positive results were sent to DNA sequencing. Results showed the speices producing KPC including KPN, ECL, CFR, KOX, ECO and EAE.84.1% of the CRE strains were KPC-2 producer. OXA-69like carbapenmase was positively detected in two isolates.We also detected?-lactamases resistance genes by PCR, including ESBLs and plasmid-mediated AmpC enzyme genes. Results showed that 74 isolates were CTX-M-type ESBLs producers. SHV-12 type ESBLs were detected in 33 isolates, accouting for 40.2%, except one SHV-31. No TEM type ESBLs gene was observed. AmpC enzymes were only detected as type ofDHA-1, totally 6 isolates, accounting for 7.3%. SBL-type enzymes, such as TEM, accounting for 35.4%(29/82). Isolates producing OXA-1, OXA-2, OXA-10 were detected in only one strain respectively. Among above 69 KPC-producers, one strain was KPC-only producer, and the rest 68 isolates of KPC-producing have also produced ESBL-type enzyme, and/or AmpC.To understand whether the KPC gene was on chromosome or plasmid in CRE strains, and the spread features, we selected 4 isolates producing several lactamase to conjugation experiment, including two Klebsiella pneumoniae and two Citrobacter freundii. Recipient bacteriums were Escherichia coli J53 (sodium azide resistance) and EC600 (rifampicin resistance). The results showed that transformants from 2 Citrobacter fi-eundii bacteria were obtained in the selective plate carrying KPC gene into E.coli J53 and EC600 respectivily. PCR detection of KPC gene were all positive for all the conjugants. Southern blot showed J53 abtained plasmid around 23kb; while EC600 abtained 3 plasmids and one is around 23kb, others above 23kb.PCR amplification showed that the detecting rate of classes?Integron was 59.7%(49/82), class?integron 4.9%(4/82), None was detected of class?integron. In 68 CRE K. pneumonia and 14 other CRE, the detecting rate of classes?Integron was 55.9%(38/68) and 78.5%(11/14), class?integron 4.4%(3/68) and 7.1%(1/14). There were 91.8%(45/49) isolates in class?integron-positive bacteria produced KPC carbapenemase and ESBL, in which Klebsiella pneumoniae accounted for 73.4% (36/49). In class?integron-positive bacteria, there were three producing KPC enzyme and all belonged to Klebsiella pneumonia. In two isolates both detected class?and?integron, both were KPC and ESBL producing Klebsiella pneumonia. Three fragments of different size were obtained after amplifying the variable region sequence of calss?integron, approximately 250bp,750bp and 1000bp. Digestion with Hinf?of variable region sequences resulted in 3 different groups of fragments. Sequencing results of variable region showed no resistance gene cassettes, except aac(3)-?gene.This set of data suggested:?KPC-2-type carbapenemases are the main cause for carbapenem resistance in Enterobacteriaceae.?KPC-2 gene could be carried by a plasmid and can be transferred between different species and spread(E.coli abtained plasmids carrying KPC from CFR).??integron strains were highly detected in this study (59.7%,49/82).Modified Hodge test and 3-amino-phenylboronic acid suppression test showed high sensitivity in Enterobacteriaceae carbapenemases detection. Statistically compared with the PCR method, modified Hodge test and 3-amino-phenylboronic acid suppression test for Enterobacteriaceae carbapenemase detection rate was not significant different.Part three:Membrane porin analysis of carbapenem-resistant Klebsiella pneumoniaeSodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of outer membrane porin showed 22 CRE isolates expressing low level of OmpK35 or its homology (eg. OmpC), of which 17 were KPN; there were 22 CRE lack of OmpK35 or its homology, of which 15 were KPN; and 38 CRE with normal expression of OmpK35 or its homology, of which 36 were KPN.10 CRE isolates expressing low level of OmpK36 or its homology (eg. OmpF), of which 5 were KPN; 57 CRE lack of OmpK36 or its homology, and 48 were KPN; 15 CRE with normal expression of OmpK36 or its homology, of which all were KPN. There were 12 CRE lack of both OmpK35 and OmpK36, of which 9 were KPN.65.8%(54/82) CRE isolates lack of at least one or two porins namely OmpK35 and OmpK36.12 CRE were expressing normally of outer membrane porin, all were KPN. Most strains were loss Ompk36, there were 48 isolates in KPN, and 9 in others.This suggested that outer membrane porin is likely to play a certain role in bacterial resistance.Part Four:Case study of patients with CRE and sensitive strainsComplete information of case history about 68 patients where CRE strains were isolated was retrospectively analysed, meanwhile, another 85 case histories of patients where carbapenem-sensitive strains were detected were selected for comparison and analysis of clinical characteristics. We first eliminated or included patients who colonized with CRE or CSE to analyze the potential infaction risk factors, then eliminated other type of strains except KPN to analyze the potential infection risk factors in patients with KPC-producing strains (non-KPC-producing set including 2 condition:CRE group with non-KPC-producing and CSE group with non-KPC-producing; CSE group with non-KPC-producing). Statistics showed that among patients with carbapenem resistant Enterobacteriaceae infection, the mean days of hospitalization was 49.55±36.91,56(96.6%) patients were hostipal-aquired infection; 2 (3.4%) were community infection.37.9%(22/58) had one or more surgery,63.8% (37/58) had one or more sickbed change,15.5%(9/58) had 3 or more than 3 times of sickbed change.56 patients had invasive operations, such as Tracheotomy, the depth of venipuncture, lumbar puncture and/or drainage and so on. About 4.8% (55/58) and 53.4%(31/58) patients using Catheter or Receiving mechanical ventilation. During hospitalization, the use of antibiotics as follows:70.7% (41/58)?35.3%(24/58)?39.7%(23/58)?43.1%(25/58) and 75.9% (44/58) patients were using carbapenems, quinolones, aminoglycosides, cephalosprorins and other classes of antibiotics(such as tetracyclines, anti-TB drugs, oxazolidinone, fosfomycin class, vancomycin, and anti-fungi drugs etc.), respectively. The average total cost of treatment among 58 patients was 4.08×106±1.74×105 yuan. In addition,6 patients died,4 patients un-cured and the rest were all improved or cured. Univariate statistic analysis of CRE infection found that hospital acquired infection, carbapenems applications, invasive operation, surgery, sickbed change, aminoglycosides application, underlying deseases, neurosurgery and ICU were showed statistically different. Multivariate analysis showed hospital acquired infection was independent risk factor for CRE infection.Univariate statistic analysis of KPC-producing carbapenem resistant Klebsiella pneumonia infection found that hospital acquired infection, carbapenems applications, invasive operation, neurosurgery and ICU were risk factors for KPC-type Klebsiella pneumoniae strains infection. Logistics analyzed that hospital acquired infection, neurosurgery and ICU were the independ risk factors for KPC-producing Klebsiella pneumoniae strains infection.This set of data suggested:?carbapenems and cephalosporins are commonly used in clinical with large amount.?hospital acquired infection was independent risk factor for CRE infection. Hospital acquired infection, neurosurgery and ICU were the independ risk factors for KPC-producing Klebsiella pneumoniae strains infection.?should strengthen the monitoring of clinical drug-resistant strains. For example bed-side isolation of patients carrying or infecting with CRE; good hand-hygiene habit; limitation of bed changing; proper use of carbapenems.Conclusion:1. CRE strains isolated from Huashan hospital were mainly Klebsiella pneumonia (82.9%), and had been detected in Escherichia coli, Citrobacter Fraundii Enterobacter aerogenes, Enterobacter cloacae, and Klebsiella oxytoca.2. Antimicrobial susceptibility test results showed that CRE were mostly pan-resistant strains, resistant to antimicrobials commonly used in clinical. In this study, the MIC90 value of CRE to imipenem, meropenem and ertapenem were 64?g/mL,128?g/mL and 256?g/mL. Colistin and tigecycline showed high vitro activity against CRE, with sensitivity of 92.7% and 68.3%, however, their clinical effection needs further investigation. |
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