| Background:Enterobacteriaceae are gram-negative bacillus-free bacteria.Most of them belong to the normal flora of the intestine and are classified as fermentative and non-fermentative bacilli.Under certain conditions(such as invasive treatment,widespread use of antibiotics,decreased body immunity,etc.),some Enterobacteriaceae bacteria can cause meningitis,pneumonia,peritonitis and cystitis,etc.,and have become the main pathogen of nosocomial infection.With the application of antibiotics,there is a large number of drug-resistant enterobacteriaceae,which seriously threatens the lives of patients.Carbapenem antibiotics produce ?-lactamases,Ampler class C ?-lactamase(AmpC),and even extended-spectrum ?-lactamases(ESBLs),Enterobacteriaceae-resistant strains cause significant infections and are used as the last line of defense against severe infections.However,with the clinical application of carbapenems such as Imipenem and Meropenem,more and more fermentative carbapenem-resistant enterobacteriaceae have been discovered at home and abroad.Carbapenem-resistant Enterobacteriaceae(CRE)has become one of the "super bacteria".According to the resistance genes they carry,CRE is divided into A type KPC,SME,NMC,IMI type;B type IMP,VIM,NDM type;D type OXA type and so on.The epidemiology and genotype research of CRE in China is mainly concentrated in the first-tier cities such as Beijing,Hangzhou,Shanghai,Xi'an and Tianjin.The resistance mechanisms of carbapenem-resistant bacteria in the regions are significantly different.It is necessary to carry out the resistance mechanism or pathogenicity of carbapenem-resistant bacteria in the northwest of Hubei Province,which is located in the core area of the South-to-North Water Diversion Project.In this paper,through the investigation of drug-resistant intestinal gut bacteria resistance in northwestern Hubei Province,carbapenem-resistant genotypes detection and pathogenicity analysis,it will provide help for clinical drug use,hospital infection control and prevention.Objective:To test the sensitivity of some fermentative Enterobacteriaceae bacteria against clinically recommended antibiotics;to analyze carbapenem-resistant genes;and to study the resistance of the Klebsiella oxytoca B1645-1 strain carrying the blaNDM-1 gene.Drug Mechanism and Its Effect on BALB/c MiceMethods:To collect blood,sputum,midstream urine,prostatic fluid,secretions,or drainage fluid from hospitalized patients in the northwestern region of Hubei in 2013.The conventional agar culture method was used to isolate Enterobacteriaceae:Klebsiella pneumoniae,Klebsiella oxytoca,Escherichia coli,Enterobacter cloacae and Serratia spp;strains were identified and tested using the Vitek-2 analysis system Resistance;PCR amplification of resistance genes to carbapenems(Imipenem and/or Meropenem)strains.Genome-wide sequencing of the K.oxytoca B1645-1 gene was performed.The resistant plasmids were extracted and transformed into E.coli DH5a.The resistance of the transformed strains was identified.Experimental BALB/c mice were intragastrically infused with K.oxytocaB1645-1 and saline at 48 h,96 h,144 h,192 h,and 240 h,respectively.The physiological,biochemical,and pathological features of the mice were observed Changes.Results:A total of 2291 isolates ofK.pneumoniae,E.coli,E.cloacae andSerratia were obtained in this study:the produce rate of ESBLs was 47.4%,62.6%,9.1%and 22.6%,respectively.A total of 46 resistant-carbapenem strains were isolated from clinical,K.pneumoniae P2964 and K.pneumoniae B195 carries blapc-2,K.pneumoniae P6558 andK.oxytoca B1645-1 carries blaNDM-1.Sequencing results showed thatK.oxytoca B1645-1 was a newly discovered K.oxytoca carrying the blaNDM-1 gene.Compared with other strains,there were significant gene insertions and deletions,as well as a small amount of inversions and translocations.Other genome rearrangement events such as tetc,cml,van,and pbp mutate by 98%,95%,80%,and 80%,respectively,and these changes are the key to bacterial resistance.Plasmid transformation showed that E,coliBDH was resistant to penicillin ampicillin(AMP)(MIC ?32),?-lactamase inhibitor ticarcillin Clavulanate T/C and ampicillin/Ampicillin sulbactam(AAM)was resistant(MIC ? 128/2;MIC=32/16)but did not show resistance to Imipenem,Meropenem properties.Animal experiments showed that after the BALB/c mice were infected with K.oxytoca B1645-1 in the stomach,the water intake in the control group was 12.5±1.6mL/2d,and the highest increase was in the high-infection group(0.12× 1010 cfu/mL).The mice rose sharply from8±1ml/2d(48 h)(P<0.05)to 16.67±1.5mL/2d(144 h)(P<0.05).By 240 hours,the drinking water of infected mice gradually returned to normal.Feed intake in BALB/c mice was5.5 ± 0.5g/2d in control group,and it increased to11.27 ± 0.4g/2d(0,12 × 1010cfu/mL,144h)after infection(P<0.05).The most obvious change was high infection.In the concentration group of bacteria(0.12 × 1010 cfu/mL),the mice with the least increase in feeding were infected with low-concentration group bacteria(0.12 × 106 cfu/mL).By 240 hours,the food intake of infected mice basically returned to normal.The change of neutrophils in BALB/c mice was small and there was no obvious rule.Bacterial counts in the cecum:BALB/c mice infected with K.oxytoca B1645-1 after stomach infection compared with controls,Enterococci at low concentrations(0.12 x 106 cfu/mL)and medium concentrations of bacteria(0.12 x 108 cfu/mL)The BALB/c mice showed a significant increase in 96 hours(P<0.05),which increased to(504.5 ± 38.9)× 106 cfu/mL and(2554.5 ± 194)x 106 cfu/mL,respectively,while the high concentration group.Enterococcus significantly reduced(69.5 ± 9.2)× 106cfu/mL,(P<0.05).At 144-240 hours,the enterococci of the infected mice showed a decreasing trend;Enterobacteriaceae appeared to 96 hours after infection.Significantly decreased(P<0.05)and basically returned to normal at 192h;Bifidobacterium compared with the control group,even dropped to<0.01 × 106cfu/mL at 48h after infection(0.12× 108 cfu/mL)(P<0.05),but it increased rapidly 10-60 times at 96 hours(P<0.05)and gradually returned to normal at 192 hours.Lactobacillus infection had little effect on the number.Pathological sections of BALB/c mice exhibited chronic inflammatory reactions in early intestine and mild inflammation in mice with low concentration of infection.Mice infected with high concentrations of bacteria exhibited chronic severe inflammatory reactions in the mid-term and all of them were late in the experiment.Rats are in recovery phase.The blood endotoxin content of the BALB/c mouse control group was about 9.49 ± 0.22pg/mL,and the endotoxin in the mouse blood was multiplied(P<0.05)after infecting bacteria.The endotoxin level of the infected mice was always at a high level.In the post-infection BALB/c mice,compared to the control group,the blood procalcitonin was transiently elevated(48 h)in addition to the high-concentration group of bacteria(0.12 x 1010 cfu/mL)(P<0.05),procalcitonin was significantly decreased at other times(P<0.05),and the procalcitonin of the infected mice was still at a low level at 240 hours.There was no obvious change in the blood C-reactive protein of BALB/c mice after infection compared with the control group.Conclusion:The resistance rate of enterobacteriaceae in some fermented regions in northwestern Hubei is on the rise and there are rare Penicillium carbacidus strains.The transmission of drug-resistance genes between strains may cause the risk of infection spread,especially among inpatients.Clinical treatment,prevention,and researchers pose challenges.K.oxytoca B1645-1 is the first strain of enterobacteriaceae carrying the blaNDM-i gene found in northwestern Hubei.K.oxytoca B1645-1 was found to be a new subspecies of Klebsiella omithinolytica in Klebsiella oxytoca by sequencing and biochemical reactions.The drug-resistant plasmids carried by K.oxytoca B1645-1 were transformed into drug-sensitive E.coli DHS?,which showed only certain penicillin-resistant characteristics(excluding carbapenems),presumably K.oxytoca B1645-1 resistant carbapenem mechanism may also be related to the bacterial chromosome or genes encoding other proteins on the plasmid.The discovery of blaNDM-1 plasmid provided a basis for the existence of resistance gene transfer among strains and provided a new idea for gene elimination of multiple drug resistance genes.At the same time,multi-drug resistant K.oxytoca B1645-1 can cause intestinal microflora dysfunction,but the normal flora of the intestine prevents the influence of drug-resistant strains.It is initially clear that intestinal microbes play a key role in eliminating inflammatory reactions. |
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