Characterization Of The β-lactamases Produced By Carbapenem-resistant Gram-negative Rods

Carbapenem antibiotics first came into clinical use in early 1990s. They have potent activity against almost all Enterobacteriaceae bacteria due to their high stability to most β-lactamases and outstanding permeability. This class of antibiotics is the most important antimicrobial agents against gram-negative rods producing ESBLs and AmpC. However, carbapenem-resistant gram-negative rods have emerged after the wide clinical use of carbapenems, which has become a serious problem all over the world.Production of carbapenemases is one of the most important mechanisms explaining the carbapenem resistance in gram-negative rods. In order to characterize the carbapenemases produced and clarify the role of these enzymes in carbapenem resistance in Huashan Hospital, a tertiary hospital in Shanghai, China, we collected all the imipenem-resistant gram-negative rods from the microbiology laboratory of Huashan Hospital from June 2003 to September 2004. The isolates we studied are Pseudomonas aeruginosa (n=79), other Pseudomonas spp. (n=13), Acinetobacter spp. (n=7), and Citrobacter freundii (n=8). We investigate their type of carbapenemases, gene location, homology, their production of other 8-lactamases and drug resistance spectrum.PART1 Detection of carbapenemases in carbapenem-resistant Gram-negative rodsAntimicrobial susceptibility testing was performed by agar dilution method. Metallo-β-lactamases (MBLs) were screened by Modified Hodge Test and EDTA-disk synergy test. The encoding genes of MBLs and oxacillinase were amplified and analyzed by PCR and DNA sequencing.Results of antimicrobial susceptibility testing showed the MICs of imipenem against 75 strains of Pseudomonas aeruginosa (75/79), 10 strains of Pseudomonas spp. (10/13), 6 strains of Acinetobacter spp. (6/7) and 8 strains of C. freundii(8/8) were ≥8mg/L. Thirteen stains of imipemem-resistant Pseudomonas aeruginosa and 3 imipemem-resistant pseudomonas spp remained meropenem susceptible, while all the imipemem-resistant Acinetobacter spp and C. freundi were resistant to meropenem at the same time. Two stains of P. aeruginosa and 2 strains of Pseudomonas putida. produced VIM-2 metollo-β-lactamase. All the 6 isolates of carbapenem-resistant Acinetobacter spp. produced OXA-23 enzyme. All the 8 strains of C. freundii produced a new subtype of IMP. The results demonstrate that the production of carbapenemases is one of the important mechanisms of carbapenem resistance in gram-negative rods. However, Carbapenemases are not the main mechanism for carbapenem-resistance in P. aeruginosa, while OXA-23 and IMP type MBL are the major cause of carbapenem resistance inAcinetobacter spp and C. freundii.PART 2 Study on β-lactamases produced by carbapenem-resistant Acinetobacter spp.Modified Hodge Test and EDTA-disk synergy test were used to screen carbapenemases in 6 carbapenem-resistant Acinetobacter strains. Isoeletronic point of the β-lactamases produced by these strains were determined by isoeletronic focusing. Encoding genes of ESBLs, AmpC, oxacillinase and metallo-β-lactamases were amplified and analyzed by PCR and DNA sequencing. Gene location was determined by Southern Blot. Strain homology was investigated by PFGE.All the 6 strains of carbapenem-resistant Acinetobacter spp. were positive by Modified Hodge Test but negative by EDTA-disk synergy test, indicating their production of carbapenemases, but not MBLs. PCR and DNA sequencing demonstrate that all the 6 strains produced OXA-23 (pI 6.9). Besides, five of the six strains produced PER-l(pI5.3), simultaneously. The OXA-23 gene is located on chromosome. Three strains of Acinetobacter junii showed identical PFGE type, while the 3 Acinetobacter baumanii isolates belonged to two different PFGE types. All the 6 strains were isolated from sputum. Clinical data analyze showed all the six patient were from ICU and had hospital acquired pneumonia. Five patients were supported with mechanical ventilation. We can, therefore, conclude that OXA-23 production is one of the molecular mechanisms of carbapenem resistance in Acinetobacter spp.The six patients were from ICU with hospital acquired pneumonia, 5 of them using mechanical ventilation support ( 3 with translaryngeal intubations, 2 with tracheotomy). Thus we conclude that OXA-23 production is one of the mechanisms of carbapenem resistance in Acinetobacter spp.PART 3Study on the β-lactamases produced by carbapenem-resistant C. freundii isolatesDuring the one year study period, we collected 8 strains of pan-drug-resistant C. freundii isolated from the urine of 8 patients. We used screening tests and isoelectric focusing to estimate the production of ESBLs and AmpC enzymes in order to clarify the mechanism of carbapenem resistance in C. freundii because carbapenem-resistant Enterobacteriaceae were rarely identified in our hospital. ESBLs, AmpC, and MBL genes were amplified and analyzed by PCR and DNA sequencing. Strain homology was investigated by PFGE. All the 8 strains were positive by both modified Hodge test and EDTA-disk synergy test, indicating theirproduction of MBLs. PCR and DNA sequencing showed that all the 8 strains of C. freundii produced TEM-1 and CTX-M-14 β lactamase. Four of 8 strains produced CTX-M-3 at the same time. No plasmid mediated AmpC was found. Furthermore, a new subtype of MBL with one amino acid different from that of IMP-8 was identified, the pI of which is about 8.4. All the 8 strains belonged to the same PFGE type. The eight patients were inpatients from Neurosurgical Department during June, 2003 to October, 2003. They all had neurosurgery on the condition of general anesthesia. Before the anesthesia they all had inserted urine catheter and later suffered catheter-related UTI. We conclude that carbapemem-resistant C. freundii produces more than one type of β-lactamases including MBL and ESBLs. All these isolates are clonally related.PART 4 Location, cloning and expression of a new subtype of IMPDuring our study period, we found a new IMP type MBL in 8 strains of carbapenem-resistant C. freundii. We have done conjugation, transformation and Southern Blot to locate the new gene. We then clone the gene into E. coli DH5a. Plasmid PUC18 was used as the vector in cloning test. Both the vector and PCR product were double digested by EcoR I and Xba I before they ligated by T4 ligase. The recombined plasmid was then transferred into E. coli DH5a. The transformants were screened by PCR. Antimicrobial susceptibility testing of 11 different antimicrobial agents on 36 strains of transformants was done to evaluate the hydrolyzing characteristics of the new enzyme. Furthermore, class 1, 2 and 3 integrons were tested to study the transmission of the gene. Results showed that the new gene couldn't be transmitted by either conjugation or transformation. Southern blot further approved that the gene encoding the new subtype of IMP was located on chromosome. All the transformants were positive in EDTA- synergy test. The enzyme is able to hydrolyze carbapenems and the third and fourth generation of cephalosporins, but has no activity on aztreonam, amikacin and ciprofloxacin. At least 3 kinds of class 1 integrons were found in the carbapenem-resistant C. freundii, but only one kind of class 1 integron contained the new IMP gene. The new gene was just at the downstream of the recombining site, which indicating its high expression level.In summary, production of carbapememases is among the main mechanisms of carbapenem-resistance in gram-negative rods, especially in Acinetobacter spp. and C. freundii. In Huashan hospital, among carbapenem-resistant gram-negative rods, VIM-2 is the main type in Pseudomonas aeruginosa and Pseudomonas spp, while a new subtype of IMP lead to carbapenem-resistant in C. freundii and OXA-23 β-lactamases lead to carbapenem-resistant in Acinetobacter spp. There were clonal transmission in Intensive Care Unit and NeurosurgicalDepartment. The strains producing carbapenemases were resistant not only to imipenem, meropenam, the third and fourth generation of cephalosporins, aztreonam, and 6-lactamase inhibitor combines, but also to aminoglycosides and fluoquinolones, which cause difficulty in antimicrobial therapy. Except the production of carbapenamases, these strains also produce ESBLs including PER-1, CTX-M-3, CTX-M-14, which may collaborate its high level antimicrobial resistance. The gene coding the new type of IMP and OXA-23 are both located on chromosome. Most β-lactams showed higher MIC to the transformants with the recombined plasmid containing the new IMP gene, but MIC of imipenem and meropenem to the transformants were lower than the clinical strains. The reason may be that the IMP gene is just at the downstream of the integron recombining site, which indicating its high expression level.