ObjectiveTo analyze the phylogroups of Klebsiella pneumoniae isolates from laboratory rats and mice housed at the Laboratory Animal Center Fudan University, optimize the identification method for Klebsiella pneumoniae, and perform virulence genes detection and antimicrobial susceptibility testing, thereby providing a practical basis for microbial detection strategies and prevention and control measures in laboratory animal facilities. MethodsBetween September 2023 and September 2025, ileocecal contents were collected from laboratory rats and mice purchased from laboratory animal suppliers, from which 11 strains of Klebsiella pneumoniae were isolated and cryopreserved. These strains were tested for β-lactamase genes using multiplex PCR for phylogrouping. Furthermore, representative strains from different phylogroups were selected for biochemical tests, mass spectrometry analysis, 16S rRNA gene and tyrB gene sequencing, as well as virulence genes detection and antimicrobial susceptibility testing. ResultsMultiplex PCR detection of β-lactamase genes revealed that among 11 strains, 4 were identified as Klebsiella pneumoniae (Kp1), 4 as Klebsiella quasipneumoniae (Kp2), and 3 as Klebsiella variicola (Kp3). Kp1-Kp3 could not be differentiated using biochemical tests, mass spectrometry analysis, or 16S rRNA gene sequencing, whereas multiplex PCR of β-lactamase genes and tyrB gene sequencing enabled phylogrouping of all three. There were differences in the detection rates of capsular genes (capsule associated gene G and magnesium transport system ATP-binding protein A) and siderophore genes (Klebsiella pneumoniae iron uptake system genes) among Kp1-Kp3; susceptibility to different antibiotics also varied considerably. ConclusionKlebsiella pneumoniae phylogroups isolated from commercially produced laboratory rats and mice are similar to those in human clinical samples and likewise include multiple phylogroups of the species complex. In this study, the identification methods of Kp1-Kp3 were optimized,furthermore selected virulence genes and antimicrobial resistance in Kp1-Kp3 were preliminarily investigated, which could provide precise reference for the treatment of infections caused by different phylogroups occurring in laboratory animal facilities.

Objective Conduct routine microbiological monitoring of laboratory rats and mice from vendors to provide an important basis for the scientific management of laboratory animal facility and ensure the reliability of relevant experimental data obtained from laboratory animals.Methods Taking the Department of Laboratory Animal Science of Fudan University as an example, between April 2021 and April 2023, rats and mice purchased from 7 vendors were sampled for microbiological quality according to the principle of simple random sampling on the arrival days of animal delivery. Then, surveillance tests were conducted to examine the microbiological contaminations according to the national standards of SPF laboratory animals.Results The total qualified rate was 80.36%, with 52.63% in SD rat, 82.76% in inbred mice, 86.67% in outbred mice and 86.36% in immunodeficient mice in details. The most frequent bacteria isolated were Staphylococcus aureus, Pseudomonas aeruginosa, Klebsilla pneumoniae and Rodentibacter heylii, and their detection rates were 10.76%, 3.16%, 2.53% and 0.63%, respectively. Serological assays demonstrated the highest prevalence for virus was Sendai virus, and the detection rate was 2.53%. In addition to the pathogens those must be excluded from SPF rodents, Entamoeba muris and Enterobacter spp. were also detected in inbred mice, and Klebsiella oxytoca was detected in immunodeficient mice, with the detection rates of 1.15%, 2.30% and 4.55%, respectively.Conclusion There are certain incidences of pathogen infections in laboratory rats and mice from vendors, and an efficient microbiological monitoring of laboratory animals should be implemented in animal facilities, in order to eliminate pathogen infections in laboratory animals, which is required for improving the accuracy of research results and protecting the occupational health of laboratory animal practitioners as well.

Objective To establish a rapid monitoring method of the three common bacteria in mice frozen resources, such as embryo, sperm, etc. Methods To extract DNA of the three positive bacteria( Staphylococcus auerus, Klebsiella?pneumoniae and β?hemolyticstreptococcus) , and establish PCR monitoring method of the three positive strains through designing primer and refining PCR condition. Then extract total DNA of the frozen resources, detect the DNA according to the PCR condition of the three positive bacteria, some samples were detect by fluorescence quantitative PCR at the same time. Results ①successfully establish a PCR detection method of the three positive bacteria, the minimum detectable concentration of Staphylococcus auerus, Klebsiella pneumoniae and β?hemolytic streptococcus is 4?19 × 10 -5 ng/μL, 1?98 × 10 -5 ng/μL and 1?07 × 10 -3 ng/μL. ②Proved that the three bacteria doesn ’ t exist in the sample by normal PCR and fluorescence quantitative PCR methods. Conclusions Establising a rapid monitoring method of common pathogens of frozen embryo and sperm in mice.

Objective To compare the effects of different transplantation sites on the outcome of testicular grafts in mice, and to provide a basis for development and application of this technique in relevant research .Method 5-day old and 4-week old SPF male C57BL/6J mice were used in this study .Three groups of testicular transplantation , i.e.dorsal subcutaneous transplantation ( 5 mice, 40 testes ) , transplantation inside the testicular tunica albuginea ( 6 mice, 12 testes), and were subrenal capsule transplantation (10 mice,15 testes) groups were set up for evaluating the effect of transplantation site on the outcomes in mice .Sham operation (4 mice) and castration (4 mice) groups were also used in this study.The mice were sacrificed at 8 weeks after transplantation and the transplanted testes were collected for analysis of their weight, transplant survival, weight gain, and germ cell differentiation.Results There were significant differences of the testicular germ cell differentiation in different transplantation groups .The germ cell differentiation was best in the in-tra-tunica albuginea transplantation group , and were similar to that in the sham operation group .The germ cell differentia-tion rate was 100%in the intra-tunica albuginea transplantation group , 29.2% in the subcutaneous transplantation group and 0%in the subrenal capsule transplantation group .Conclusions Transplantation beneath the testicular tunica albug-inea is the most favorable site for germ cell differentiation , and dorsal subcutaneous transplantation is an alternative choice . Subrenal capsule transplantation is not appropriate for preservation of male reproductive organs in mice .

Objective To compare the effect of three cryoprotectants on vitrification-cryopreservation of C57BL/6J mouse epididymis.Method Epididymises from 6-8-week old male C57BL/6J mice (age) were cryopreserved using DMSO, PROH and R18S3 cryoprotectant solution and thawed , respectively.The morphology of sperm from thawed epididy-mis, the rate of post-thawing survival and reproductive capacity were determined to evaluate the freezing efficiency of the three cryoprotectants .Results The sperms from thawed epididymis of the three groups were all well-preserved structural-ly.The survival rate of sperms was 88.17%, 61.17% and 16.83% in the PROH, DMSO and R18S3 cryoprotectant solu-tions, respectively, and there were significant differences between the three cryopreservation groups (P<0.05 for all). The number of pups from the DMSO , PROH and R18S3 groups were 13, 8 and 17 mice after ICSI and embryo implantation , respectively.Conclusions All the three cryoprotectants DMSO , PROH and R18S3 solutions are suitable for vitrification-cryopreservation of C57BL/6J mouse epididymis.But PROH is the preference of these cryoprotectant solutions .