Enterobacter cloacae AmpC enzyme detection of high-yield and
molecular epidemiological study
Author: Huda Chun Shao Jian-chun Yang Shaomin Li
Chao Ping \
【Abstract】 Objective To study the hospital Enterobacter cloacae AmpC enzyme and molecular epidemiological
characteristics of high-yield. Methods The KB method
susceptibility testing, double disk synergy test and enzyme extract high-yielding three-dimensional test for detection of
Enterobacter cloacae AmpC enzyme, polymerase chain reaction
for detection of AmpC enzyme, ERIC repeat rep PCR, Research
Kunming First People's the hospital in July 2002 ~ December 2004 of 84 clinical isolates of Enterobacter cloacae resistant, AmpC enzyme genotype and clone spread of conditions. Results ESBLs phenotype screening test and confirmatory test, high-AmpC enzyme detection rate of 27.38% (23/84), ESBLs detection rate of 16.67% (14/84), while high-
yield AmpC enzymes and ESBLs detection rate of 44.05% ( 37/84). AmpC gene detection rate of 43.75% (28/64). AmpC-
dimensional test yielding a detection rate of 47.62% (40/84). Cloning communication analysis and found that strain No. EC2 and EC11, EC13 and EC14, EC19 and EC20, respectively have the same DNA fingerprint and the remaining no DNA fingerprints
between strains. Conclusion Enterobacter cloacae resistance is more complicated. Go out to deter the performance of high-
yielding AmpC enzyme, producing ESBLs, simultaneous production of AmpC enzymes and ESBLs and plasmid AmpC enzyme multi-drug-
resistant phenotype. Molecular epidemiology revealed that, although the spread of Enterobacter cloacae and antibiotic induction and selection of the role of resistance in patients with decreased body and gut dwelling bacteria infection of endogenous factors, but still want to guard against the
occurrence of hospital infection.
Key words Enterobacter cloacae AmpC β lactamase enzyme
extracts from three-dimensional test re-order sequence primed
polymerase chain reaction (ERIC rep PCR)
Gene detection and the molecular epidemiology of Enterobacter cloacae
with hyperproduction of AmpC β lactamase
ABSTRACT Objective To investigate the molecular epidemiology of Enterobacter cloacae with hyperproduction of AmpC β lactamase. Methods Using the Kirby Bauer (KB)
antibiotic susceptibility test, cloxacillin synergistic
disc diffusion and the modified three dimensional
extraction test, E.cloacae hyperproducing AmpC β lactamase
were examined. The differential genotypes of AmpC β
lactamase were investigated using the Enterobacter repetitive intergenic consensus (ERIC) for repetitive extragenic palindromic element based PCR (rep PCR). The study was
carried on in the First People's Hospital of Kunming from July, 2002 to December, 2004 in 84 clinically separate cases of E.cloacae. We determined the drug resistance profile,
genotype and molecular epidemiology of each strain. Results Using phenotypic screening and extended spectrum β
lactamases (ESBLs ) confirmatory test, twenty three strains
(23/84, 27.38%) of E.cloacae were found to have hyperproduction of AmpC β lactamase. The rate of ESBLs
positive samples was 16.67% (14/84) and 44.05% had elevated AmpC β lactamase and ESBLs (37/84). The rate of AmpC β
lactamase gene detection was 43.75% (28/64). Using the three
dimensional extract test, we observed AmpC β lactamase
detection rate of 47.62% ( 40/84). Through molecular epidemiology analysis, the results showed that the strains EC2 and EC11, EC13 and EC14, EC19 and EC20, had the same genotypic trees respectively. The rest of the strains did not exhibit this trait. Conclusion The drug resistance profile of E.cloacae is quite complex. The strains that expressed hyperproduction of AmpC β lactamase only, ESBLs only, or
both at the same time, demonstratedvariable drug resistance between them. The results of the molecular epidemiological research demonstrated that even the transmission of E.cloacae resistance was often induced by the improper therapeutic
option of drug, but also related to the decrease of the autoimmune condition of the patients and the enteric endogenous infections, whereas vigilant attention to the incidence of community / hospital acquired infections couldn't be ignored.
KEY WORDS Enterobacter cloacae; AmpC β lactamase;
Modified three dimensional extract test; ERIC rep PCR
In recent years, cephalosporins widely used in China and its effect on the choice of the role of Enterobacter cloacae, Enterobacter cloacae producing ESBLs  and the AmpC enzyme
 has become the clinical focus of attention. Ma Yue such reports , Enterobacter cloacae clinical resistance to antibiotics commonly used rate, in addition to imipenem and meropenem, the eight-year period in varying degrees of growth.
Ceftriaxone, cefotaxime, ceftazidime, ciprofloxacin, gentamicin and amikacin resistance to antibiotics, such as rate increased by 20% or more. In this study, phenotypic screening tests, double disk synergy test and enzyme extract three-dimensional test, detection of infection from clinical specimens isolated from 84 high-yielding Enterobacter cloacae
AmpC enzymes and ESBLs, while polymerase chain reaction for detection of AmpC enzymes genes, with the repeat sequence between Enterobacter (Enterobacter repetitive intergenic
consensus, ERIC) primers were amplified and cloned dissemination of research status report on the results as follows.
1 Materials and methods
1.1 Source and identification of strains
Collected in July 2002 ~ December 2004 in Kunming First
People's Hospital in-patient and out-patient infection in
patients with various types of censorship Enterobacter cloacae isolated from specimens of 84, excluding parts of the same patient to repeat the same isolates. Sputum specimens from 68
of them (80.95%), throat swab 3 (3.57%), urine samples four (4.76%), purulent secretions 5 (5.95%), cerebrospinal fluid 1 (1.19%), nosocomial infection surveillance samples 3 (3.57%). Specimens mainly from the hospital ICU (40%), neurosurgery (22%), and veteran cadres Section (16%). In accordance with the "national clinical laboratory operating procedures,"
second edition of the regular train separation using BD BBL Crystal ENF strain identification system identified to species.
1.2 AmpC enzyme phenotype screening
Using CLSI / NCCLS (1999 years) recommended by Kirby
Bauer disk diffusion method, the Guangdong Yue Tongtai offers French Bio Merieux Company Mueller Hinton agar, the
United Kingdom Oxoid sensitivity produced paper, imipenem (IMP), cefepime (CPE), ceftriaxone (CRO), cefotaxime (CTX), ceftazidime (CAZ), cefoxitin (CFX), cefotaxime / clavulanic acid (CD03, 30μg/10μg), ceftazidime / clavulanic acid (CD02, 30μg/10μg). CFX, and AMPC-resistant as a screening indicator . Then IMP, CPE, CD02, CD03, CRO 5 Zhong paper to further
distinguish . The results showed that, CPE, IMP performance sensitivity, CRO, CD02 and CD03 or their antimicrobial resistance ring memory in a few colonies, in order to deter the continued high-yield AmpC enzyme, suggesting that IMP-
sensitive rest were resistant to judge for the high-yield AmpC
enzymes and ESBLs exist.
1.3 cefoxitin three-dimensional test
Using modified enzyme extract three-dimensional
(1) Preparation of β -lactamase crude extract to 35 ?
overnight culture of test bacteria produced 1.5 × 108CFU/ml
bacilli, take 50μl by adding 12ml of tryptone broth. Purchase 35 ? constant temperature shaking bed (200r/min) incubated for 4 ~ 6h, 3000r/min centrifugal 25min, discard supernatant.
Precipitate obtained by adding 0.01mol / L of PBS (pH7.4) 1.0m1, vortex mixing, and placing -170 ? and 37 ? liquid
nitrogen repeated freezing and thawing 5 times, 4 ?
12000r/min centrifugal 1h, the supernatant enzyme extract shall be material. Prepared to take a good enzyme extract agar plate was inoculated into normal, 35 ? incubated for 18 ~
24h, confirmed that no bacterial growth, -25 ? to save.
(2) three-dimensional test (indirect method) to E. coli ATCC25922 by KB France coating MH agar, in its central paste
CFX (30μg) paper, using sterile blades 5mm away from the paper at the edge of a radial cut slit, take 25 ~ 40μl enzyme
extract by adding slit, the home to be a little dry 35 ? 18 ~
24h. If slit and Antimicrobial ring appears to expand at the
junction of the long strain of regional, sub-contractors for
the AmpC-positive, indicating AmpC enzymes tested bacteria for high-yield strains of Enterobacter cloacae 029 as a negative control, E. cloacae 029M (People's Liberation Army 301 Hospital, Microbiology Branch gift) as a positive control.
1.4 ESBLs Detection
Screening and confirmation of ESBLs tested according to CLSI / NCCLS (1999 years) recommended method, CTX ? 27mm, CAZ
? 25mm, CRO ? 25mm, ATM ? 27mm 4 pieces of paper there are
two or more smaller than the above-mentioned criteria for
screening positive for a suspicious strains, the need for confirmation tests. CD02 and CAZ, CD03 and the difference between the diameter of CTX antibacterial ? 5mm, identified
as positive for ESBLs.
AmpC-producing Enterobacter cloacae 1.5 Genotyping
(1) Bacterial DNA extracted using alkaline lysis method, kit (ACT 1, MIR 1 and DHA 1, DHA 2) from Wuxi
Institute of Genetic Technology. Taking into colonies containing 100μl normal saline 0.5ml centrifuge tubes,
centrifuged (15000r/min) 5min, discard supernatant, add lysis buffer A 50μl, lysis buffer B 2μl, blending into 55 ? heat
after 1h, and then set into the 95 ? heat 5min, centrifuged
(10000r/min) 30s, the supernatant liquid shall be amplified
(2) PCR amplification reaction according to kit instructions, take 5μl template solution by adding 15μl of
heat pipe in heat two drops of paraffin oil can be affixed.
(3) PCR amplification conditions of 93 ? predegeneration
2min, then 93 ? 30s ? 55 ? 20s ? 72 ? 60s a total
circulation of 35 cycles, final extension of 72 ? to 2min.
PCR products obtained 10μl with 2μl bromophenol blue
indicator mixing, point-like in 2% agarose gel in order to 100V voltage electrophoresis 20 ~ 30min, emergence and the
positive template molecule, the same band (302bp) for the ACT
1, MIR 1-positive. DHA 1DHA 2 bands of 405bp.
1.6 repeat sequence primed polymerase chain reaction (rep
(1) The source of bacterial strains of DNA extraction,
the main instruments are ibid.
(2) The oligonucleotide primers Enterobacter gene duplication sequence (ERIC)  as: ERIC1 5 '
ATGTAAGCTCCTGGGG ATTCAC 3', ERIC2 5 ' AAGTAAGTGACTGGGG
TGAGCG 3', from Shanghai Balboa Biotechnology Limited
synthesis. Reposted elsewhere in the paper for free download http://
(3) The reagent Taq enzyme, dNTP, MgCl2, 10 × PCR Buffer
is owned by Shanghai Boya Biotechnology Co., Ltd. products. DNA molecular size Marker for Fermentas LIFE SCIENCE PRODUCTS:
GeneRulerTM 100bp DNA Ladder Plus, molecular size range of 100 ~ 3000bp. Agarose for the REGULAR products.
(4) PCR amplification reaction with reference  carried out. 10 × Buffer 2.5μl, Mg2 50mmol / L 1.25μl, dNTP (10mmol
/ L) 0.5μl, Taq DNA polymerase 2.0U, primers (50pmol) 0.5μl,
sterile deionized water 14.75μl, samples of DNA 5μl, the
total volume of 25μl .
(5) PCR amplification conditions for the light of literature  the design, pre-denaturation 95 ? 7min,
denaturation 90 ? 30s, annealing 52 ? 1min, extension 65 ?
8min, thermal cycles 30, the final degeneration of 90 ? 30s,
annealing 52 ? 1min and extending 65 ? 16min.
(6) PCR detection of amplified products with a 1.5% agarose gel electrophoresis separation of PCR amplified
products, ethidium bromide (EB) staining, Gel Doc 2000TM gel imaging analysis system analysis DNA bands, and use the software Quantity One right rep PCR fingerprints (band) for
bacterial homologous analysis.
2.1 phenotypic screening tests had confirmed the Experimental high-yield AmpC enzyme, yields ESBLs,
simultaneous production of AmpC enzymes and ESBLs detection rates were 27.38% (23/84), 16.67% (14/84), 44.05% (37/84) ;
11.9% (10/84) strains of AmpC enzymes, ESBLs were negative.
AmpC enzymes and the same time, a simple high-yield production
of two enzymes AmpC enzymes and ESBLs strains accounted for 71.43% (60/84).
2.2 cefoxitin three-dimensional test
AmpC-positive 40, ie close to the direction of the side
of the paper cefoxitin apparent decrease of the formation of bacteriostatic ring notch, the positive rate was 47.62% (40/84), in which AmpC-phenotype screening test detected a simple high-yield and the simultaneous production of AmpC enzyme AmpC enzymes and ESBLs two kinds of enzymes of 60 strains of bacteria have 38 three-dimensional test AmpC-
positive, accounting for 63.33% (38/60), 14 pairs of paper synergy test detected ESBLs bacteria yields in 2-D test
positive for AmpC enzyme, accounting for 14.29% (2 / 14).
2.3 AmpC enzyme gene
AmpC gene detection rate of 43.75% (28/64). 64 Enterobacter cloacae AmpC enzyme positive results of genetic testing and phenotype screening method and three-dimensional
test positive results compared in Table 1.
2.4 The status of cloning coverage
Of 64 strains of Enterobacter cloacae to rep PCR
analysis and found that strain No. EC2 and EC11, EC13 and EC14, EC19 and EC20, respectively have the same DNA fingerprint; remaining between strains no DNA fingerprinting.
Table 1 64 Enterobacter cloacae phenotypic screening method and three-dimensional test, AmpC enzyme gene analysis of test results
AmpC β lactamases can be found in chromosomal or plasmid . Chromosomal AmpC-inducible and continuing into
high-yielding type; and plasmid AmpC enzyme with or without inducing agent, are sustainable mass production [10,11], but also often carry multiple drug resistance genes such as aminoglycosides, chloramphenicol , fluoroquinolones and other
types of β lactamases (such as ESBLs) and so on, so that
the growing complexity of drug resistance. In this study, paper screening method, confirmatory test and three-
dimensional test for detection of the 84 clinical isolates of Enterobacter cloacae resistant phenotype, mutation detection to deter high-yielding strains of AmpC enzyme 23 (27.38%), yields ESBLs 14 strains (16.67 %), simultaneous production of AmpC enzymes and ESBLs 37 strains (44.05%). Beijing Gu Yi-ming
 the results were 65.32%, 13.79% and 20.69%, Xian Liu's results , respectively 42.48%, 26.55% and 9.73%, Wuhan Jin-Ling Shi  the results of 16.0%, 10.4% and 13.2%. The results of this study show that the First People's Hospital of Kunming, Enterobacter cloacae clinical isolates of AmpC enzyme derepression mutation detection rate of high-yielding lower
than in Beijing and Xi'an, higher than the Wuhan; yield results with ESBLs closer to Gu Yi-Ming; simultaneous
production of AmpC - and the ESBLs over domestic reports,
analysis for two reasons: ? with the main source of the
specimens. Specimens of this study come mainly from the hospital ICU (40%), neurosurgery (22%), and veteran cadres Section (16%); ? the clinical use of antibiotics because of drug-resistant strains of the differences in habits and lead to regional differences in drug resistance genes . Our results show that high-yield AmpC enzymes and ESBLs-resistant
Enterobacter cloacae have become two major factors. Enterobacter cloacae AmpC enzymes and ESBLs produced at the
same time, the drug resistance is extremely serious, with the exception of imipenem, the resistance to almost all antibiotics.
Three-dimensional test AmpC-positive rate was 47.62%
(40/84). Although the enzyme extract three-dimensional
experiment can be detected AmpC β lactamases, but can not
distinguish between chromosome or plasmid mediated. One reason for the type of plasmid AmpC enzymes are more active for the hydrolysis of cefoxitin different, and second, the amount of enzyme extract also affect test results.
Analysis of 64 Enterobacter cloacae AmpC enzyme gene test results. Phenotypic testing for mutations in order to deter the 12 high-yielding AmpC-positive strains have seven AmpC
enzyme (blaACT 1, blaMIR 1 and / or blaDHA 1DHA 2)
amplification-positive, accounting for 58.33%; 12 ESBLs yields only an AmpC enzyme gene positive (8.33%); 34 simultaneous production of AmpC enzymes and ESBLs in AmpC enzyme gene
positive strain rate 44.12% (15/34). Enterobacter cloacae
resistant phenotype showed more complex, laboratory detection of drug resistance still needs to be improved and improved.
Repeat fragment gene amplification by PCR amplification of bacterial DNA fragments to obtain the strain of repeated-
specific genome. Enterobacter repetitive intergenic sequence (ERIC) length of 126bp, located on chromosome non-coding
transcribed spacer, including a highly conserved central inverted repeat region and at the bacterial genome outside the region, due to different bacteria ERIC location of the gene is different from Therefore, a better classification results, with a resolution higher than random primer PCR, the efficiency of ribosome sub-type good . EC2 and EC11, EC13
and EC14, EC19 and EC20, respectively have the same DNA
fingerprints, showing characteristics of vertical transmission. ICU clinical data show the same as the source of specimens, EC2 and EC11 No. November 2002, January 2003 specimens; EC13, EC14 was in February 2003 specimens; EC19, EC20 No. March 2004, April specimen, there are clear time consistency. Tips should strengthen the hospital infection control, cut off a variety of transmission routes.
There are 90.63% (58/64) of Enterobacter cloacae seen the same DNA fingerprints, indicating that the spread of
Enterobacter cloacae and the extensive use of cephalosporins and other broad-spectrum β lactam antibiotics in the
induction and selection, the a serious underlying diseases, receive invasive treatment methods, such as catheter devices, body resistance in patients with decreased intestinal bacteria dwelling endogenous infection. Rational use of antibiotics, development and use high-tech means of simple, rapid and
accurate monitoring of drug resistance is urgent need to strengthen research and solve problems.
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