Detection And Genotypes Distribution Of Extended-spectrum β-lactamases In Proteus Spp.

Proteus spp. are Gram-Negative bacillus which are generally distributed in the environment, such as in polluted water, soil and rubbish, and also in intestinal tract of human and animal. They are conditional pathogenic bacteria, usually causing secondary infection, such as chronic otitis media, wound infection, cystitis, infantile diarrhea and alimentary toxicosis. Proteus spp includes Proteus vulgaris, Proteus mirabilis, Proteus myxofaciens and Proteus penneri. Among them, Proteus vulgaris and Proteus mirabilis are clinically relevant species. The mechanism of drug resistance of Proteus spp to β -lactam antibiotics mainly is due to the production of β -lactamase, especially Extended Spectrum β -lactamases (ESBLs).ESBLs are defined as plasmid-mediated β -lactamases capable of hydrolyzing oxyimino β -lactam antibiotics that are inhibited by clavulanic acid (CA), such as tert-cephalosporin. ESBLs are produced mainly by Gram-Negative bacillus, which are divided into five groups: TEM, SHV, CTX-M, OXA and other type. Most of the ESBLs are resulted from either single or several point mutations from TEM-1, TEM-2 and SHV-1. The plasmid encoding drug resistance gene can be horizontally transmitted, which therefore can easily cause ourbreak of nosocomial infections. The problem of bacterium ESBLs resistance is one of the significant global nosocomial resistance problems.Since TEM type of ESBL was first reported in 1994 in France, ESBLs-producing Proteus spp have become more and more prevalent strains in the world. The major resistant gene types are CTX-M and TEM. During the past few years, the rate ofESBLs production in other species of Enterobacteriaceae was gradually increased. In 1997, a study was carried out by Saurina on Enterobacteriaceae drug resisitance in 15 hospitals in Brooklyn, New York. Nine point five percent (9.5%) of Proteus spp were found to produce ESBLs, which was lower than that of Klebsiella pneumoniae(44%), but higher than that of Escherichia coli (4.7%).We collected 45 Proteus spp. strains from 1^stAffiliated Hospital of WenzhouMedical College and 2^nd Affiliated Hospital of Zhejiang University to analyze theresistance and detect the Extended-spectrum β-lactamases genotypes of β -lactamantibiotics resistant strains.Methods:1. Forty five (45) Proteus spp strains were cultured and identified by morphological analysis and by VITEK automatic identification system.2. Minimum inhibitory concentration (MIC) of 45 Proteus spp strains was determined by E-test.3. Extended-spectrum β -lactamases was detected by preliminary screening test, disc confirmatory test and three dimensional test.4. Conjugation of ESBLs phenotype positive strains was performed using E. coli EC600 as receptor.5. Isoelectric focusing electrophoresis was used to detect Isoelectric points of the ESBLs phenotype positive strains.6. Primers of TEM, SHV, CTX-M were synthesized, and genes of ESBLs were amplified by Polymerase chain reaction (PCR) and the PCR products were analyzed by Sequencing.Results:1. Forty two (42) isolates were susceptible to clinical used common antibiotics including tert-cephalosporin, and their 50% MIC and 90% MIC values were very low. Other three Proteus spp strains were resistant against multiple antibiotics including tert-cephalosporin.2. There are three ESBL_S phenotype positive strains (9,30 and 38).3. Plasmid conjugation of three ESBL_S phenotype positive strains (9,30 and 38) wassuccessful, and the resistance of transconjugants resulted from the isolates were slightly lower than their parent strains.4. Genes were amplified by PCR among the three strains and sequenced, As a result of blasting in Genbank, the genotypes of ESBL_S are CTX-M-14 and TEM-1, and there is no SHV and other genotypes of ESBL in these three isolates. Isoelectric points of CTX-M-14 and TEM-1 are 7.9 and 5.4.Conclusions:1. Most Proteus spp. isolates were susceptible to clinical used common antibiotics including tert-cephalosporin and their 50% MIC and 90% MIC values were very low. Three ESBL_S-producing Proteus spp were detected. Conjugation studies showed that ESBLs genes were encoded on conjugative plasmids which can cause horizontal transmission of drug resistance genes.2. CTX-M-14 are the main genotypes of ESBLs in Proteus spp. from the two hospitals.