This study attempts to develop a reference substance for the live bacteria count of Streptococcicosis live vaccines in order to evaluate the validity of live bacterial count in inspection and testing. We prepared a batch of live Streptococcus suis reference substance for live bacterial count, tested their physical property, purity, vacuum degree, remaining moisture, and determined their homogeneity, thermal stability and transportation stability. Moreover, we organized collaborative calibration to assign count values to the reference substance and determine the shelf life of the reference substance in 12 months. The results showed that the physical property, the purity, the remaining moisture and the vacuum degree of the reference substance were all in compliance with the requirements of the Chinese Veterinary Pharmacopoeia. The homogeneity test showed that the coefficient of variation of the count of the reference substance was less than 10%, indicating a good homogeneity. Transportation stability test showed that the reference substance remained active after 72 h transportation in summer and winter with the package of styrofoam boxes and ice packs. Thermal stability test showed that the reference substance could be stored for up to 3 months at -20 °C, or up to 21 days at 4 °C. According to the collaborative calibration, the reference vaccine was assigned a count value range of (8.5-12.1)×107 CFU/ampoule. The shelf life test showed that the reference substance was stable for 12 months when stored at -70 °C. The reference substance could provide a reference for the live bacterial count of Streptococcicosis live vaccines. Moreover, it could also be used as a reference to evaluate the quality of corresponding agar media.
Bacterial Load ; Reference Standards ; Vaccines, Attenuated

Objective To evaluate the performance of modified Hodge test on the detection of carbapenemases among Enterobacteriaceae with decreased susceptibility to carbapenems. Methods Fortynine Enterobacteriaceae isolates with decreased susceptibility to carbapenems ( MIC of imipenem, meropenem or ertapenem was ≥ 2 μg/ml ) were collected from 16 teaching hospitals from 2004 to 2008. MICs of imipenem, meropenem and etapenem were determined by agar dilution method. Carbapenemases were detected by modified Hodge test. Carbepenemase-causing positive results and AmpCs-causing positive results were differentiated by phenyl boronic acid and oxacillin. Beta-lactamases encoding genes including blaNDM-1were detected by PCR and sequencing. Results Thirty-six of 49 isolates were non-susceptible to imipenem (MIC >4 μg/ml), 31 were non-susceptible to meropenem (MIC > 4 μg/ml) and 47 were non-susceptible to ertapenem (MIC > 2 μg/ml). Twenty-three isolates showed positive modified Hodge test result, including 9 weak-positive results and 14 strong-positive results. Through PCR detection and sequencing, 2 out of 9 isolates showing weak-positive results carried blaKPC-2 and other 7 did not carry any carbapenemase genes but AmpCs/ESBLs genes. Among the 14 isolates showing strong-positive results, 4 carried blaKPC-2, 8 carried blaIMP-4 and 2 caried blaIMP-8. All 26 isolates with negative modified Hodge test result didn't carry any carbapenemase genes. No isolate carried blaNDM-1. Carbapenemases genes PCR detection was regarded as a gold standard, and the sensitivity, specificity, positive predictive value and negative predictive value of modified hodge test was 100%, 79%, 70% and 100% on the detection of carbapenemases among Enterobacteriaceae with decreased susceptibility to carbapenems. Conclusions Modified Hodge test revealed great sensitivity but showed a few false positive results. True and false positive results can be effectively differentiated by phynel boronic acid and oxacillin.

Objective To evaluate the influences of susceptibility interpretation of Escherichia coli,Klebsiella pneumonia and Proteus mirabilis in China mainland according to the old and new ceftazidime,cefotaxime and ceftriaxone breakpoints in CLSI M100-S20 and CLSI M100-S19. Methods First, We analyzed the antibacterial susceptibility results of the three bacteria by agar dilution method in the SEANIR surveillance item, which were collected from 15 national hospitals between the year of 2005 and 2007 and excluded the AmpC enzyme positive isolates according to the PGR-DNA sequencing method and/or the antibacterial susceptibility phenotype. ESBL phenotype was confirmed by the CLSI phenotypic confirmatory test. Antibacterial susceptibility of the total 2733 Escherichia coli, Klebsiella pneumonia, Proteus mirabilis isolates was retrospectively analyzed by WHONET 5. 4 software according to the breakpoints of the CLSI M100-S19 (S19) and CLSI M100-S20 (S20). Second, 207 isolates of Peking Union Medical College Hospital with the results of both agar dilution method and disk diffusion method were performed by recurrent analysis. Then we observed the inter-method agreement through the scatter diagram according to the breakpoints of S19 and S20. Results First, as to the ESBL positive Escherichia coli, Klebsiella pneumonia and Proteus mirabili.s, the resistant rate of cefotaxime increased from 65.2% , 55.5%, 14. 6% under S19 (64 μg/ml) to 99. 7%, 96. 2% , 93. 8% under S20 (4 μg/ml). The susceptibility rates decreased from 6. 0%, 11.5%, 33.3% under S19 (8 μg/ml) to 0%, 0. 2%, 0% under S20 ( 1 μg/ml). Ceftriaxone had the same trend as cefotaxime. Though ceftazidime was more active than cefotaxime and ceftriaxone, as to the ESBL positive Escherichia coli and Klebsiella pneumonia, the resistant rates slightly increased from 30. 3%,43. 2% under S19 (32 μg/ml) to42.0%, 56. 0% under S20 (16 μg/ml). The susceptibility rates slightly decreased from 58. 1%, 44. 1% under S19 (8 μg/ml) to 44. 7%, 28.0% under S20 (4 μg/ml). Second,as to the ESBL negative Escherichia coli, Klebsiella pneumonia and Proteus mirabilis, all the susceptibility rates of ceftazidime, cefotaxime and ceftriaxone were between 99. 2%-100. 0%, the resistant rate were between 0%-0. 4%. Third, the S20 MIC breakpoints had a good correspondence with the ESBL phenotype.Fourth, according to the recurrent analysis of MIC testing and disk dilution method, r value was 0. 67,0. 79, 0. 77 for ceftazidime, cefotaxime and ceftriaxone, respectively, and all P value were under 0. 01. The intermethod rates of S19 and S20 were both acceptable. Conclusions If the cefotaxime and ceftriaxone S20 new breakpoints were used, the concordance of antibacterial susceptibility results and ESBL phenotype would increase greatly. The clinician could select proper antibiotics according to the antibacterial susceptibility results and clinical symptoms. It is no longer necessary to edit results for cephalosporins, aztreonam, or penicillins from susceptible to resistant. However, until laboratories implement the new interpretive criteria,ESBL testing should be performed as described in Supplemental Table 2A-S1. The relationship between the new breakpoints of ceftazidime and clinical outcomes need to be further evaluated.

OBJECTIVE To observe the bacterial biofilm formation on indwelling double J stent in the human body.METHODS A prospective study of 42 cases with double J stent indwelling in the human body after operation was conducted.The stent was divided into 4 segments then detected microbiologicaly in a separate manner and observed with scanning electron microscope for the presence of bacterial biofilm.RESULTS From 42 cases with douple J stent,only one case was without any bacteria in four segments.20 cases(47.6%) had the same bacteria in four segments.The bacterial positive rate of each segment was pelvic segment(81.0%),upper ureter segment(64.3%),lower ureter segment(71.4%) and bladder segment(83.3%);totally 18 kinds and 126 bacterial strains were flund.the most popular pathogens were Escherichia coli(23.0%),Pseudomonas aeruginosa(15.9%),corynebacterium(15.1%) and Staphylococcus epidermidis(13.5%).On the surface of unused double J stent,there was uneven but clean,without any attachment and encrustation,but on the surface of double J stent indwelling in the human body,the inflammatory affachment and encrustation were observed as well as bacterial biofilm,encapsulated in the abundant fibrous membrane.CONCLUSIONS Our study found that bacterial biofilm can be encrustated on the surface of double J stent indwelling in the human body,it may be one of the reasons for catheter-associated urinary tract infection.