| Cronobacter spp. is considered as an opportunistic bacterium. Infections are oftenreported in neonates, premature infants and immune-compromised infants. It can causemeningitis, necrotizing enterocolitis and septicaema with fatality rates more than40%.Although the sources of Cronobacter spp. pollution in nature is unclear, the a lot of casesinvolved in outbreaks and sporadic of Cronobacter spp. infection in infants have showed thatcontamination of powdered infant formula (PIF) is strongly involved in this infection. So, it isparticularly important for research the prevalence of Cronobacter spp. and characteristics ofthe isolates. In this study, contamination rules of isolates of Cronobacter spp. had beeninvestigated in key points in representative three goat milk powder processing factories in inShaanxi province, and studied virulence genes, antibiotic susceptibility, thermal treatment,chemical fungicide resistance and the molecular subtypes of the Cronobacter spp. isolates bypulse field gel electrophoresis (PFGE). The main conclusions are as follows:1. Among760samples collected from different production processes in factory A, B andC.67(8.8%) samples were positive for Cronobacter spp., the prevalence rates to samples infactory A, B and C were12.1%(31/256),12.3%(32/261) and1.6%(4/243), respectively. Inthe67isolates,24(35.8%) isolates are mucus shape and43(64.2%) isolates are smooth.Powder samples had shown the highest positive rate (28.4%), among which the positive rateof floor powder, soil, intermediate products and final products. Swabs samples (10.9%),plants internal air samples (6.7%) and liquid samples (3.2%) also detected Cronobacter spp..Samples of floor powder and swabs had a higher detection rate.2.67isolates identified by PCR. The results were as follows.1) Isolates of C. sakazakii(88.1%) were the predominant species of Cronobacter spp., followed by C. malonaticus(9.0%), C. muytjensii (1.5%) and C. universalis (1.5%). No C. turicensis, C. condimenti and C.dublinensis isolate was found. Virulence genes cpa, hly. sip and ompX of67isolates weredetected.2) The rate of virulence gene was100%. The detection rates of virulence genotypescarrying ompX, ompX+hly, ompX+cpa and ompX+cpa+hly were16.4%,3.0%,37.3%and43.3%, respectively. No sip gene was found.3. Antimicrobial susceptibility test of isolates showed that the highest rates of resistant was rifampicin (100%), then followed by trimethoprim/sulfamethoxazole (49.3%). Allisolates were100%sensitive to ciprofloxacin, gatifloxacin, gentamicin, streptomycin,ampicillin, ceftriaxone, cefoperazone and tetracycline. The resistant rates to differentantimicrobial of isolates detected from three factories were different. While resistant rates ofrifampicin and trimethoprim/sulfamethoxazole were consistent. Multiple resistant rates (≥3)of Cronobacter spp. was7.5%.4. Results of thermal treatment showed that the D60of smooth isolates were0.77~0.95min and the D60of mucus shape isolates were1.22~1.54min which weresignificantly greater than that of smooth isolates (P<0.05). The isolates of Cronobacter spp.were inactivated in short time at the temperature above80°C. The results of chemicalfungicide resistance were that three mucus shape isolates were alive by the treatment of1.5%and2.0%HNO3after15min. All strains were inactivation by the treatment of1.5%,2.0%,2.5%NaOH,3%and5%bleaching powder,3%H2O2and75%alcohol. It was indicated thatacid resistance of isolates was stronger than that of alkali. Meanwhile, the resistance ofthermal and acid treatment of mucus shape isolates was stronger than smooth isolates.5. Sixty seven Cronobacter spp. isolates were categorized into27different PFGEclusters after PFGE with XbaI. Forty three of67isolates were grouped into five main clusters(named as D-1to D-5). All PFGE clusters of the isolates were unique within each factory, andsubstantial genetic homogeneity of the isolates was observed among the different samplinglocations in the same factory. Thirty one isolates recovered from samples collected in factoryA were grouped into11clusters. Substantial genetic homogeneity was observed amongisolates belonging to intermediate products and various environmental settings (spray drying,fluidized bed, floor powder of fluidized bed rooms, transmission belt, shoes, floor and floorpowder of packaging rooms). It was indicated that floor powder of plants and personalcross-infection were potential dissemination routes of contamination in intermediate products.Thirty two isolates recovered from factory B were grouped into12clusters. Substantialgenetic homogeneity was observed among isolates belonging to drain, spray drying and spraydrying platform. Four isolates recovered from factory C were grouped into4clusters andshowed lower genetic homogeneity of a similarity coefficient below5.0%. |
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