Concentration And Transmission Of Microbiological Aerosol And Molecular Identification Of Indicator Bacterial Resistance In Hospital Surroundings

Microorganisms are the main factors of acquired infection in hospitals. Microorganisms in bioaerosol from hospital houses can cause serious air pollution. They may also affect the health and the production capability of the patients and induce prevalence of aerosol infectious diseases. The polluted air in hospital wards or Out Patient Departent (OPD) is often associated with the outbreak of the epidemic diseases and the environmental problems. It is known that many airborne pathogenic microorganisms, including viruses and bacteria, can spread over a large area through the air. Bioaerosol disseminated from wards to their environments has been studied with an emphasis on total bacterium amount, pathogenic bacteria and antibiotic resistances of the bacteria in hospital wards and their ambient air. It is difficult to differentiate between two strains that have very close genetic relationship using traditional bacterial taxonomy, and can not get the enough evidence to prove the transmission of microorganism from hospital houses to their surroundings. So, air samples, including wards ( the E. coli strains isolated from corrider at 5m or 10m to ICU ward away) and OPD air of 5 ICU wards in 5 different hospitals and their corriders, 3 injection rooms of the OPD and their corriders,hall were collected using six-stage Andersen microbial samplers and RCS samplers. E. coli and Staphylococcus aureus concentrations (CFU/m3 air) collected from different sampling sites were calculated. In order to study microbiological aerosol spreading from hospital wards to their surrounding air, the following methods were used: firstly, airborne aerobic bacteria concentrations collected from different sampling sites were calculated; secondly, the antibiotic-resistance and resistant genes of 87 strains of E. coli were detected; thirdly, the enterobacterial repetitive intergenic consensus polymerise chain reaction (ERIC-PCR) was used to study the genetic variability and to determine the strain relationships among E. coli isolated from different sites in each hospital wards or injection room of the OPD; fourthly, the serotyping of 131 strains of E.coli was identified using 4 groups of pathgenic factors; finally, the heterogeneity and resistance and bacteria genome fingerprinting were detected combining flow cytometer technique with RFLP (Restriction Fragment Length Polymorphism) and PFGE ( Plused-Field Gel Electrophoresis.The bacterial apoptosis of E.coli was also detected applying PI and AnnexinⅤdouble staining.Depended on these tests, the hazardness of bioaerosols and transmission to hospital environment have been confirmed.1. Concentration of airborne aerobe bacteria, Escherichia coli and Staphylococcus aureus in ICU and OPD injection roomThis experiment used the ANDERSEN-6 air collector and RCS samplers to collect air samples in five different sampling points (Rooms A, B, C, corridor, and hall) in the Injection Room of the Out Patient Department (OPD) of a city-level hospital in Shandong, China. By detecting the concentration of airborne aerobic bacteria, Escherichia coli and Staphylococcus aureus in hospital environment as well as their distributing to each stage on ANDERSEN-6 sampler, the harm which possibly caused to the patient and medical affairs staff can be deduced and arising the people attach great importance to the bioaerosol in hospital environment.The results indicated that the concentration of mean airborne aerobes and respirable airborne aerobes were 126.8 and 70.38 CFU/m3 (47.9-70.2%) in 5 sites, respectively. The differences among airborne aerobes and respirable airborne aerobes were no significant differences (p>0.05) in 5 sites.The overall average concentrations of total airborne E. coli and airborne Staphylococcus aureus were 63 and 96 CFU/m3, and respirable airborne E. coli and airborne Staphylococcus aureus were 43 and 81 CFU/m3 in swine houses, respectively. Furthermore, there were highly significant differences in concentrations between the total airborne E. coli and airborne Staphylococcus aureus in each ICU (p<0.01).The ratios of respirable airborne Staphylococcus aureus to the total concentrations were ranged from 59.8% to 79.2% in 5 sites, respectively, but both there were no significant differences among the five sites (p>0.05). Airborne aerobes and Escherichia coli in the five sampling points were 211 and 18 CFU/m3; 206 and 18 CFU/m3; 304 and 13 CFU/m3; 567 and 22 CFU/m3; and 651 and 18 CFU/ m3, respectively. The respective concentrations of aerobes and inhalable aerobes in the corridor and hall were apparently higher than those in the rooms (p<0.05). The airborne aerobes were mainly Staphylococcus and Escherichia. Gram-negative bacteria accounted for 40.08%~46.98% of the total aerobes.2. Transmission identification of the Escherichia coli aerosol in hospital environment to their surroundings using ERIC-PCRAir samples, including five ICU rooms (Room A, B, C, corridor, and hall) and one injection room were collected using six-stage Andersen samplers and RCS. E. coli concentrations (CFU/m3air) collected from different sampling sites were calculated. Furthermore, the enterobacterial repetitive intergenic consensus (ERIC)-PCR method was applied to amplify the isolated E. coli strain DNA samples. Through the genetic similarity analyses of the E. coli obtained from different sampling sites, the spreading of bioaerosol from hospital environment to the ambient air was characterized. The results showed that 48 strains of the E. coli isolated from indoor air had 100% similarity, and 22 strains had 90% similarity with those isolated from outdoor (5 m, 10 m) in five ICU wards;10 strains of the E. coli isolated from three injection rooms had 100% similarity, and 18 strains had 90% similarity with those isolated from the corridor, the hall. Our results suggested that some strains isolated from outdoor air originated from indoor air. This also could be concluded that microbiological aerosols in indoor air of hospital could transmit to their surroundings via air exchange and cause microbiological contamination.3. Serotyping of E. coli of air environment in hospitalsFour groups of pathogenic factors of 131 strains of E. coli separated from the out-patient injection rooms and the ICU wards and air environment around were further identified at the same time by using serological methods. On the one hand, it can verify a variety of infantile diarrhea caused by E.coli which results cross-infection in hospital whether it is through the air spread in the hospital environment;On the another hand, whether it exists O111, O55, O26, O119, O127, O157:H7 and O128 and other serogroups in 131 strains of E. coli ,which have been identified as infant diarrhea agentsin many countries around the world. The results show that, by serological agglutination test, 27 strains of EPEC were positive, they are a total of seven kinds of serotype. The dominant serotype O142: K86 (B) are 12 strains, three strains of O26: 60 (B6), 3 strains of O127a: K63 (B8), 3 strains of O44: K74 (L), 3 strains of O114: K90 (B), 2 strains of O125: K70 (B15), 1 strain of O111: K58 (B4); There are 10 positive ETEC, three kinds of serological type, of which six strains of O25: K19 (L), 2 strains of O8: K40 (A) K47, 2 strains of O78: K80; nine strains of EIEC are agglutinate, there are two kinds of serum type, of which five strains of O112ac:K66, four srtains of O124: K72; the O157: H7 serum type can not be checkouted.4. Identification of the antimicrobial resistance and ESBLs gene of Escherichia coli in hospital environmentsAirborne E. coli was spread from indoor to outdoor via air exchange by using the method of analyzing the antimicrobial resistance of 87 E. coli isolated from the 5 sites which can cause the ambient air outside of the hospital polluted and further threaten the neighboring patients and the medical affairs staff's health. Meanwhile, theβ-Lactamase antimicrobial gene (TEM,SHV,CTX-M) of 87 E. coli were detected. The study showed that E. coli had various degrees of drug resistance to the penicillin-type drugs, antibiotics with enzyme inhibitor, carbapenem antibiotics, cephalosporin antibiotics of the first to the fourth generation, monocylic antibiotics, aminoglycoside antibiotics and quinolones drugs.The result suggested that the penicillin-type drugs had the higest drug resistant, antibiotics with enzyme inhibitor and carbapenem antibiotics had the higest sensitive rate. Through homology comparation with ERIC-PCR, the strains whose homology were up to 100% had the same resistant spectrum. This further proved airborne E. coli in hospital environment could come through the air and cause iatric cross-infection. The ESBLs antimicrobial resistance genes profile of 87 E. coli. predominate in TEM gene. According to the drug sensitive test,the E. coli. had high resistance rates, a lot of multiple drug-resistant strains and broad resistance spectrum. All above these agreed with the daily use of drugs in hospital which was known when sampling. That's showed the produce of drug tolerance may have a connection with complex drug- using situation in therapeutic procedure, the variety of drugs, unreasonable use of drugs and the resistance mechanism of E. coli.5. Identification of the antimicrobial resistant heterogeneity and apoptosis of airborne E.coli by FCMThis experiment used FCM (flow cytometer) to do the antimicrobial susceptibility testing of isolated airborne E.coli and Standard strain 25922 incubating with ampicillin or ampicillin; bacterial apoptosis was also tested by two fluorescent staining of PI (propidium iodide) and AnnexinⅤ; in addition, it was tried out using three methods: RFLP, PFGE and FCM on E.coli. The results showed that antimicrobial susceptibility testings were accurately obtained by calculating their MIC after straining DNA of E.coli with PI; a hypodiploid peak (apoptotic peak) whose area stands for DNA content showed up before G0/G1 peak and from this the apoptotic cells of all cells were calculated.Using double fluorescent staining of PI (propidium iodide) and AnnexinⅤ, FCM (flow cytometer) can distinguish normal cell, early-stage apoptotic cell, end-stage apoptotic cell and necrosis cell; Combined technology of RFLP and PFGE, FCM (flow cytometer) can not only test the drug resistance and drug resistant heterogeneity of airborne E.coli quickly and high-sensitivityly, but also establish fingerprinting DNA of E.coli, find new subtype of new strain, this can open up better technique platform to the microorganism field.