Genotypes Of Extented Spectrum Bata-lactamases And Molecular Characteristics Of Integrons Among Chicken Isolates

ABSTRACT With the widespread use of antimicrobial agents for prophylaxis and therapy of infected animals, the wild dissemination of antimicrobial resistance, especially multidrug-resistance (MDR), is increasing among chicken Enterobacteriaceae in the worldwide. There are many ways for bacteria to obtain multidrug-resistance, example for extended-spectrumβ-lactamases (ESBLs) and integrons. Therefore, the present study was delineated to identify and characterize.1. Sixty-four chicken Enterobacteriaceae isolates were identified with the Vitek-32 automatic microbe system, which were isolated from different chicken farms, Henan province. All isolates were as follows: Escherichia coli (n=51), Salmonella (n=8), Proteus mirabilis (n=4), and Acinetobacter baumannii (n=1). To our knowledge, Acinetobacter baumannii was isolated firstly from chickens. The MDR profiles of 64 clinical isolates were determined at the same time using the CLSI microbroth dilution method. The antimicrobial agents containedβ-lactam antibiotics, aminoglycosides, tetracyclines, amphenicols, fluoroquinolones, and sulfanilamide. The results demonstrated that 58.8%~74.5% E.coli showed resistance to third generation cephalosporins, 96% (49/51) were resistant to more than four antimicrobial agents, 92.2% were more than five, and 82.4% were more than six. The MDR profiles of 8 Salmonella isolates were more than four. The other isolates were sensitive to third generation cephalosporins, and their MDR profiles were not more than three.2. The ESBLs detection showed that 54.7% (35/64) isolates were ESBL-producing bacteria. With the PCR, BlaTEM was detected in 38 isolates, blaCTX-M was detected in 19 isolates and 21 isolates harboured blaOXA among 64 strains. Except for one isolate that had TEM-57, which was first detected in chicken E.coli, most of blaTEM isolates possessed TEM-1. The blaCTX-M isolates encoding CTX-M-65, CTX-M-14, CTX-M-24, and CTX-M-90 were 14, 1, 3, and 1 among 19 CTX-M-ESBL isolates, respectively. The CTX-M-90 gene was the new subtype in all ESBL genotypes. Based on the amino acid sequences of four CTX-M subgenes, we postulated possible evolution scheme was CTX-M-14→CTX-M-24 or CTX-M-90→CTX-M-65. The blaOXA contained OXA-1 (11 strains), OXA-2 (2 strains), and OXA-10 (16 strains). These findings indicated that TEM-1 was the most frequently encountered blaTEM allele in chicken clinical bacterial populations, Henan. Then the more commonβ-lactamase alleles were OXA-10, CTX-M-65, and OXA-1.3. Twenty-six out of 35 ESBL-producing isolates (74.3%) carried more than two bla genes, whereas, only 6.9% (2/29) non-ESBL-producing isolates harboured two bla genes. The resistant rates of 35 ESBL-producing isolates to ampicillin, third generation cephalosporins, and third generation cephalosporins/beta-lactamase inhibitors were 100%, 77.1%~91.4%, and 0~37.1%. The MDR profiles of those isolates were more than six, and 94.3% (33/35) were more than seven. But, the resistant rates of 29 non-ESBL-producing isolates toβ-lactam antibiotics were lower than 45% (except ampicillin), only 44.8% (13/29) owned more than six MDR profiles, and 20.7%(6/29)were more than seven. The MDR profiles of 47.4% CTX-M-ESBL isolates (9/19) were not less than ten, yet that was only 18.8% (3/16) among non-CTX-M-ESBL isolates. At the same time, the results showed that 60% isolates harbouring blaCTX-M-65 or blaCTX-M-90 had more than ten MDR profiles, 20% (3/15) had more than twelve, but the other blaCTX-M isolates demonstrated less than ten. The resulte suggested that the CTX-M-65 and CTX-M-90 subgroups were more prone to exhibit the MDR phenotypes than the -14 and CTX-M-24, indicating that there has identity of the blaCTX-M evolutionary tendency and the MDR phenotypes extended trend.4. Five non-ESBL chicken E.coli and O78 strains were cultivating in broth, which contained ceftriaxone or cefotaxime with 1/2MIC subinhibitory concentrations. When the strains were cultivated to the tenth generation, the MICs of two drugs increased 8~64 times. The MICs increased 8~64 times again and the O78 strain induced by cefotaxime expressed resistance to cefotaxime when cultivated to the twentieth generation. The strains passaged to the thirtieth generation demonstrated resistance to ceftriaxone, cefotaxime, gentamicins, amikacin, doxycycline, florfenicol, and enrofloxacin, indicating that the speed of bacteria obtaining MDR genes and presenting MDR phenotypes was fast with antimicrobial agents selective pressures.5. With PCR and sequencing analysis, O78 strain harboured TEM-1 When the strain was induced to the 20th passage with cefotaxime, the nucleotide sequence occurred one base mutation (24T→C). It had two base mutations (24T→C, 40C→T) at the 30th generation, and the latter mutation led to a new amino acid substitution (14Pro→Ser), that is, TEM-1 subtype evolved to a new subtype. This result illustrated that the antimicrobial agents selective pressures play an important role in blaTEM evolations. All strains did not carry any blaCTX-M before inductions. The O78 strain had changed to a CTX-M-14- ESBL strain When it was cultured to the twentieth generation with cefotaxime. At the thirtieth generation, All the strains had evolved to CTX-M-ESBL strains (CTX-M-90 or CTX-M-65). This finding demonstrated that the variable speed of blaCTX-M was higher than that of blaTEM. We could mainly concluded the evolutionary process of blaCTX-M by analysis the different CTX-M amino acid sequences. With the drugs selective pressures, the non-ESBL strains firstly obtained blaCTX-M-14, then blaCTX-M-90, and then blaCTX-M-65. It is worth noting that CTX-M subtypes obtained from inductive trials are detected simultaneously in chicken isolates, which suggested that the blaCTX-M evolutionary process could partially reflect that of isolates.6. Forty-four of the 51 chicken E.coli (86.3%) detected class 1 integron and 3.9% (2/51) contained class 2 integron. Five gene arrangements have been detected among class 1 integrons, containing sat (100%), dfrA1/aadA1 (45.4%), dfrA17/aadA5 (22.5%), dfrA1/sat/aadA2 (6.8%), and 4800 bp unknown. cassette array. To our knowledge, dfrA1/sat/aadA2 was a novel gene cassette array. blaCTX-M was detected in 4800 bp unknown cassette array and located the downstream by nested PCR and PCR mapping. Class 2 integrons contained the one array gene cassette of dfrA1/sat1/aadA1.7. The results showed that 93.6% ESBL-producing chicken E.coli (29/31) carried integrons, but the value was 75% among non-ESBL strains. The difference between two groups has a statistical significance, which illustrated that the ESBL-producing E.coli are more prone to harbour integrons. Among 15 non-ESBL strains with integrons, 90% isolates (12/15) carried only an integron, and the variable region only integrated one resistant gene cassette. But 89.6% ESBL strains with integrons (26/29) contained 2~4 integrons.