Inhibitory Effects Of Pseudomonas Aeruginosa Upon Other Microorganisms And Mechanism

Part I Susceptibility testing and epidemiological analysis of Pseudomonas aeruginosa[Objective]To explore sensitivity of Pseudomonas aeruginosa against antimicrobial drugs and provide a scientific basis for the rational use of antibiotics and infection control.[Method]Sixty non-repetitive strains of Pseudomonas aeruginosa were collected from clinical specimens in our hospital from June to August,2012, and were identified by Gram staining, oxidase test and API20NE system (bioMerieux, Inc, France); Drug susceptibility test was carried out by Kirby-bauer (KB) method, and clinical characteristics of the distribution and drug resistance were analyzed. All the strains were sequenced by multilocus sequence typing (MLST) using seven house-keeping genes:acsA,aroE,guaA,mutL,nuoD,ppsA and trpE. The sequencing results were analyzed based on Pseudomonas aeruginosa international MLST database to obtain the allelic profiles and sequence types(STs). MLST data were analyzed by Bionumerics4.0(http://pubmlst.org/mlstanalyse) using Splits Tree and eBurst to obtain the further genetic characterics of the strains. SPSS was used to analyze ST-type relationship between the sections of the distribution and drug resistance.[Result]Sixty strains of Pseudomonas aeruginosa possessed high drug resistance rates, and resistance rates in descending order are:minocycline (MH)(100.0%), piperacillin (PIP)(55.0%), levofloxacin (LEV)(51.6%), imipenem (IPM)(48.3%), gentamicin (GN)(48.3%), cefoperazone (CFP)(48.3%), cefoperazone/sulbactam (SCF)(46.6%), meropenem (MEM)(46.6%), ciprofloxacin (CIP)(45.0%), piperacillin/tazobactam (TZP)(45.0%), cefepime (FEP)(43.3%), aztreonam (ATM)(43.3%), amikacin (AK)(38.3%) and ceftazidime (CAZ)(15.0%). Out of the60strains,9were resistant to all antibiotics. Pseudomonas aeruginosa MLST display that60of them can be divided into11STs, such as:ST262, ST195, ST767, ST274, ST527, ST849, ST639, ST871, ST244, ST636and ST645type, with no discovery of new types; among them, ST244and ST274were the dominant STs, with ST244accounting for26.67%(16/60) and ST274accounting for18.33%(11/60). The resistance rate of these two STs was significantly higher than other types and higer in ICU and in Respiratory Medicine department than other clinical departments. It is possible that there are small-scale epidemic of nosocomial infections in our hospital.[Conclusion]Drug resistance of sixty strains of Pseudomonas aeruginosa was serious, and there were many multi-drug resistant (MDR) and pan-drug resistant (PDR) strains of them. ST244and ST274were the dominant STs in our region. There was a certain correlation between these two STs and drug resistance rate, which of them are higher than that of other STs, and there was a small-scale epidemic of nosocomical infections in our hospital. MLST is significant for Pseudomonas aeruginosa genomic study and epidemiology surveillance. Part II Observation on killing and inhibitory effect of Pseudomonas aeruginosa against other microorganisms[Objective]To observe inhibitory effect of the clinical isolates of Pseudomonas aeruginosa against other microorganisms, and to provide clues for development of anti-infective drugs.[Method]Effect of60strains of Pseudomonas aeruginosa against staphylococcus aureus ATCC25923was observed by sterilizing filter paper-disk method. We screened out two strains PA1206and PA1246which have a strong effect, and two strains PA1201and PA1222which show no effect at all. Further study of the effect against other microorganisms were focused on these four strains and observed by paper-disk method, cross-streaking method and co-culture method. The indicator microorganisms included:gram-positive cocci(Staphylococcus aureus, Staphylococcus epidermidis, staphylococcus capitis etc.), gram-positive bacilli (Bacillus, Corynebacterium, etc.), gram-negative bacilli (Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Acinetobacter baumannii etc.), gram-negative diplococcus (Moraxella catarrhalis etc.) and fungi(Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida krusei, Cryptococcus neoformans etc). We observe influence of the temperature, pH, different nitrogen and carbohydrate sources, the incubation time on the inhibition of Pseudomonas aeruginosa and observe inhibitory effect of Pseudomonas aeruginosa against staphylococcus aureus and Candida albicans in vivo through co-infection model in mice.[Results]The results show that these two strains PA1206and PA1246have significant killing effect on gram-positive cocci, such as methicillin-resistant Staphylococcus aureus (MRSA) and Vancomycin-intermediate Staphylococcus aureus (VISA), obvious inhibitory effect upon fungi and gram-negative diplococcus etc, but no inhibition on gram-negative bacilli. PA1201and PA1222show no effect on those microorganisms. Influencing factors:after incubation of24hours, cultures of PA1206and PA1246were placed in temperature between4℃to121℃and they still have a inhibitory effect on staphylococcus aureus ATCC25923. The optimal pH is at pH7, and at pH=4and pH=10, the inhibitory effects had lost. When Maltose and soluble starch as the carbohydrate source, the cultures showed relatively less inhibitory effect; when the others as carbohydrate sources, the culture showed a strong inhibory effect. When L-asparagine, ammonium phosphate as nitrogen sources, they completely suppressed the inhibitory effect of PA1206and PA1246. Ammonium nitrate, sodium nitrate can inhibit part of the inhibitory effect of PA1206and PA1246. When potassium nitrate, ammonium sulfate, ammonium chloride as nitrogen sources, PA1206and PA1246showed the strongest inhibitory effect. Incubation time displayed, from the first day to the12th day, the inhibitory effect gradually increased, and since the12th day, with the nutrient depletion, inhibitory effect waned. Co-infection models showed that Pseudomonas aeruginosa has inhibitory effect in vivo.[Conclusion]Some Pseudomonas aeruginosa stains can inhibit gram-positive cocci, gram-negative diplococcus and yeast-like fungi except gram-negative bacilli. Some of them have no effect. Various changes in environmental conditions can affect the killing and inhibitory effect of Pseudomonas aeruginosa. Pseudomonas aeruginosa has inhibitory effect against other microorganisms in vivo. Co-infection models showed that Pseudomonas aeruginosa has inhibitory effect in vivo. Part Ⅲ Inhibitory mechanism of Pseudomonas aeruginosa against other microorganisms[Objective]To explore the mechanism of inhibitory effect of Pseudomonas aeruginosa against other microorganism, and to find other ways of treating clinical infectiou diseases.[Method]Pulsed-field gel electrophoresis (PFGE) was used to analyse these four Pseudomonas aeruginosa (PA1201,1206,1222,1246) homology. Polyacrylamide gel electrophoresis (PAGE) was used to tell the difference of these four strains in secretive proteins. And then the metabolites of the four strains were extracted by chloroform and the extracts were analyzed by thin layer chromatography (TLC) and bioautography, analytical high performance liquid chromatography (HPLC) systems. The crude exracts were separated by silica gel chromatography and preparative HPLC and every peak elute was collected to obtain effective eluent composition, and analysed its molecular weight by mass spectrometry.[Results]PFGE displayed these four strains had a different homology. SDS-PAGE displayed four strains differ in the secreted proteins. Metabolites produced by PA1206and PA1246can kill and inhibit Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) and Vancomycin-intermediate Staphylococcus aureus (VISA),gram-negative diplococcus and fungi but not gram-negative bacilli. Chloroform crude extracts displayed that PA1201and PA1222were green, PA1206and PA1246color were dark brown. PA1206and PA1246presented the similar patterns and PA1201and PA1222displayed ths similar patterns by analytical HPLC, but two types of patterns are definitely different. Preparative HPLC and mass spectrometry showed that PA PA1206and PA1246produced four kinds of small molecular substance to kill or inhibit other organisms, but PA1201and PA1222produced no antibiotics, which perhaps is the reason of no effect against other microorganisms.[Conclusion]PA1206and PA1246can produce small molecular substance or secretive proteins to kill or inhibit other microorganisms. There is a posiiblity that these two strains active metabolites may be made into bactericides and applied in the treatment of clinical infections such MRSA and VISA or fungal infections.