Serratia marcescens - Term Paper Example

S. marcescens is susceptible to expanded-spectrum cephalosporins, such as ceftoxamine, ceftazidime, cefepime, and cefpirome, though different mechanisms of resistance to expanded-spectrum cephalosporins have been reported. A constitutive overproduction of Amp-C, as a result of mutation in the regulatory gene system of Amp-C expression results in resistance to expanded-spectrum cephalosporins. The other enzymes that confer resistance to expanded-spectrum cephalosporins are Ambler Class A extended-spectrum ?

-lactamases (ESBLs) or Class B metallo-?-lactamases. Mutation in chromosomal amp C gene also adds to the resistance pattern to expanded–spectrum cephalosporins in the Family Enterobacteriaceae. Enterobacter cloacae GC1, S marcescens GN16694 and S. marcescens HD are among the few clinical isolates producing extended-spectrum ?- Amp C ?-lactamases. Yatsuyanagi et.al., (2006), in their study examined the resistance mechanism of cetazidime-resistant S. marcescens strains from inpatients of a cerebral ward over a 14 month period in one hospital in Japan.

One environmental and five E.coli transformants from clinical isolates harbouring the amp-C gene, cloned from S .marcescens and E.coli AS22-6-51, which is an amp-D mutant of E.coli C600, has a deletion mutation in AmpC. The cloning vector used was pBcSK+. The MICs of azetreonam, imipenem, meropenem, gentamicin, minocycline and levofloxacin were determined by broth dilution technique. The reference strain used for in vitro susceptibility testing was E. coli ATCC 25922. Chromosomal DNA was prepared from S .

marcescens and the isolates were examined by PCR for the presence of the following genes : blaTEM , blaSHV, bla CTX-M, and plasmid borne amp-C genes. Primers specific for S.marcescens isolates were used to amplify a 552-bp fragment. Chromosomal DNA embedded in agarose plugs were prepared for Pulse-Field Gel Electrophoresis (PFGE). The primers amp-C Seq 5 was used to amplify 1.174 bp fragment containing the chromosomal amp C gene and Mab/F and MAb/R primers were used to amplify the 1.192 bp fragment containing the blaTEM gene.

These primers were used to sequence the chromosomal amp C gene and blaTEM gene from S. marcescens isolates. The primer amp C Exp5ER was used to amplify the chromosomal gene from S. marcescens strains ES46, ES76, and SM4 by PCR. Cloning of S. marcescens chromosomal amp C gene and construction of amp C harbouring E.coli transformants were carried out. Amp-C expressing transformants were confirmed to be positive for S.marcescens chromosomal amp C gene using PCR with a set of Sma amp C F and Sma amp C R primers and amp C Exp5ER and ampC Exp 3 HND primers for the 1,158 bp fragment containing the amp C gene using PCR.

A primer was used to introduce a point mutation leading to a substitution of a third motif of the amp C gene, in a PCR based site -directed mutagenesis performed withal PCR in vitro. Using overnight cultures of S. marcescens, amp C ?-lactamases induction and enzyme assay were done using an ultrasonic disrupter and the protein content was evaluated by BCA protein assay reagent. DNA sequence data analysis were performed using an updated version of Basic Local Alignment Search Tool. The nucleotide sequences of the chromosomal amp C genes of S.

marcescens were deposited in the Gene Bank data base. Results of the study showed that four strains of S.marcescens (ES11, ES31, ES42, and ES46) isolated from urine specimens showed an identical SpeI PFGE pattern, indicating that a