Performance of a novel fluorogenic probe assay for the detection of extended-spectrum-ß-lactamase or plasmid AmpC ß-lactamase-producing Enterobacterales directly from simulated blood culture bottles.

Resistance to third generation cephalosporins is widely disseminated in Enterobacteriaceae mainly because of extended-spectrum-ß-lactamases (ESBL), plasmid AmpC ß-lactamases (PABL), and hyper-production of chromosomal AmpC ß-lactamases. Here, we evaluated the performance of rapid test using novel fluorogenic probe assay in simulated blood cultures and compared the results with the phenol red assay using a total of 172 characterized isolates (39 ESBL producers, 13 PABL producers, and 120 susceptible isolates). We prepared a pellet by centrifugation and washing, which can also be used for identification with MALDI-TOF directly from positive blood cultures. After that, we mixed the pellet with fluorogenic probe and measured the fluorescent signal using fluorometer. The fluorogenic probe assay showed higher sensitivity than the phenol red assay (96.2% vs. 71.2%, p < .0001) in 172 simulated blood culture bottles especially in detecting PABL (84.6% vs. 0%, p = .0026) and the turnaround time was 1.5 h. This fluorogenic probe assay, combined with the direct identification of pathogens, could be very useful for rapid identification of isolates and detecting cephalosporin resistance caused by ESBL and PABL directly from positive blood cultures.

Phylogenetic characterization of norovirus strains detected from sporadic gastroenteritis in Seoul during 2014-2016.

BACKGROUND: Phylogenetic analysis of norovirus (NoV) is efficient for tracking NoV transmission. To determine the widespread NoV strains in Seoul, we conducted an extensive phylogenetic characterization of NoV-positives from 1659 diarrheal specimens collected in 2014-2016 for the Seoul NoV-surveillance. RESULTS: When the large numbers of NoV partial VP1 genome sequences were analyzed in acute gastroenteritis patients along with the phylogenetic characterization, we could identify molecular epidemiologic patterns based on the genetic characteristics of sporadic NoV strains circulating in Seoul, which could provide a detailed description of the genome-wide and community-wide NoV evolution in each genotype. The average NoV detection rate in our study period was 16.34% that was increased by 7.44% from 13.17% in 2014 to 20.61% in 2016. Prevalence of NoV GI and GII was 4.43% and 93.36%, respectively, and the GII.4, GII.17, and GII.3 were found to be the major type among 17 genotypes of NoV. The most prevalent one was GII.4 (50.92%) that was followed by GII.17 (18.08%) and GII.3 (9.96%). According to an extensive phylogenetic analysis based on partial VP1 sequences of 1008 NoV (276 sporadic, 518 outbreak and 214 reference), pandemic strains of GII.17, GII.4 and GII.3 have emerged in succession during the 2014-2016 Seoul NoV-surveillance. GII.17 emerged as GII.17|Kawasaki323 in 2014, and became the predominant genotype in 2015 with GII.17|2014_Kawasaki lineages (CUHK-NS-616/Kawasaki308). The formerly predominant GII.4 remained high-level with GII.4|2012_Sydney in 2014 and internally replaced to GII.4|2016_Kawasaki194 lineage (NOR-2565/NOR-2558/OH16002) that caused the sporadic NoV explosion since December 2015. Sporadically prevalent GII.3|Hu/Aichio334-13/2013 failed to develop any outbreaks, whereas sporadic GII.3|Hu/3-28/2015/HNZZ/CHN caused heavy outbreaks in Seoul without preparation time since November 2016. CONCLUSIONS: This is the first extensive phylogenetic study revealing the important events of NoV strains circulating in Seoul. Particularly, our study period from 2014 to 2016 was very dynamic with the emergences of the three main NoV strains (GII.17|2014_Kawasaki, GII.4|2016_Kawasaki194 and GII.3|Hu/3-28/2015/HNZZ/CHN) every year. We are sure that it is hard to detect above findings by simple conventional analysis. Our present study reports a future paradigm of the NoV molecular epidemiology, which might be highly valuable to track new strains and predict oncoming outbreaks.

Performance of a novel fluorogenic chimeric analog for the detection of third-generation cephalosporin resistant bacteria.

Resistance to third generation cephalosporins is widely disseminated in Enterobacteriaceae mainly due to extended-spectrum-ß-lactamases, plasmid AmpC ß-lactamases, and hyperproduction of chromosomal AmpC ß-lactamases. Here we evaluated the performance of a novel fluorogenic probe rapid test and compared the results with the phenol red assay using a total of 77 characterized organisms (44 extended-spectrum-ß-lactamases, 33 chromosomal or plasmid AmpC ß-lactamases) and 46 susceptible organisms. The fluorescent assay showed higher sensitivity than the phenol red assay in cefotaximase type extended-spectrum-ß-lactamases, non- cefotaximase type extended-spectrum-ß-lactamases, chromosomal AmpC ß-lactamases, and plasmid AmpC ß-lactamases (96.7% vs. 90.0%, p=0.157; 71.4% vs. 7.1%, p=0.003; 100.0% vs. 64.7%, p<0.001; 100.0% vs. 6.3%, p<0.001). The fluorescent assay had a positive correlation with the exponents of cefotaxime and ceftazidime minimum inhibitory concentrations (p<0.001 for both). The new fluorescent assay will be very useful for the rapid detection of resistance to third generation cephalosporins that originates from various ß-lactamases.

Prevalence and characteristics of lactose non-fermenting Escherichia coli in urinary isolates.

Recently, serotype O75 was found to be prominent among the non-ST131 ciprofloxacin-resistant Escherichia coli, and they were all lactose non-fermenters. In this study, we investigated the prevalence and characteristics of lactose non-fermenters in urinary isolates of E. coli. A total of 167 E. coli isolates was collected. Antimicrobial susceptibility tests were determined by VITEK 2 (bioMerieux, France). The lactose non-fermenters underwent PCR-based O typing, multilocus sequence typing (MLST) analysis, phylogenetic grouping. For ciprofloxacin-resistant isolates, the resistance mechanisms were investigated. Thirty-three (19.7%) isolates were lactose non-fermenters and the ciprofloxacin resistance rate was significantly higher than in lactose fermenters (66.7% vs. 31.6%, P = 0.0002). According to the serotype, O75 was the most common (42.4%, 14/33) and was followed by O16 (5/33), O2 (4/33), O25b (3/33), O15 (1/33), O6 (1/33), O1 (1/33). All the O75 isolates were ciprofloxacin-resistant and belonged to ST1193. By MLST, they were resolved into 11 STs. ST1193 was the most common (14/33) and was followed by ST131 (8/33). Interestingly, 8 isolates of ST131 were divided into three O types [O16 (4 isolates), O25b (3), and non-typeable (1)]. The ciprofloxacin resistance rate was high in isolates of O75-ST1193 and O25b-ST131 but low in O16-ST131 and O2-ST95. All the ciprofloxacin-resistant isolates showed identical triple mutations in gyrA and parC but the serotype O25b isolates had an additional mutation in parC (E84V). Only one isolate harbored aac(6')-Ib-cr variant and no qnr gene was detected. Continuous monitoring of the prevalence and clonal composition of the lactose non-fermenters is needed.

Evaluation of MicroScan WalkAway and Vitek 2 for determination of the susceptibility of extended-spectrum ß-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolates to cefepime, cefotaxime and ceftazidime.

OBJECTIVES: The aim of this study was to evaluate the accuracies of these automated susceptibility test systems with cefepime, cefotaxime and ceftazidime using the new CLSI and EUCAST guidelines in extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae. METHODS: A total of 220 ESBL-producing clinical isolates were collected from 12 hospitals in Korea. Susceptibility testing for cefepime, cefotaxime and ceftazidime was performed by MicroScan WalkAway, Vitek 2 and the CLSI broth microdilution test. ESBL genotypes were determined by PCR amplification. RESULTS: The proportion of isolates classified as susceptible to cefepime and ceftazidime with the CLSI and EUCAST guidelines was 35.0% versus 2.3% for cefepime (P < 0.001) and 21.8% versus 8.2% for ceftazidime (P < 0.001), respectively, and the susceptible isolates were mainly the CTX-M-9 group or SHV-type ESBL producers. All of the isolates were resistant to cefotaxime. Against the total of 220 ESBL-producing isolates, using the CLSI (EUCAST) criteria, very major/major error rates of MicroScan and Vitek 2 were as follows: 1.9%/20.8% (1.8%/20.0%) and 27.4%/0% (12.2%/0%) for cefepime and 2.6%/8.3% (1.2%/0%) and 4.5%/0% (2.3%/0%) for ceftazidime, respectively. The very major error rates of MicroScan and Vitek 2 with cefotaxime were 0.9% and 1.4%, respectively. The errors were mainly major errors for MicroScan and very major errors for Vitek 2. CONCLUSIONS: A substantial portion of ESBL-producing isolates were susceptible to cefepime and ceftazidime by using the CLSI and EUCAST breakpoints. Unfortunately, the error rates of the two automated susceptibility systems were not acceptable for cefepime and ceftazidime.

Prevalence and mechanisms of decreased susceptibility to carbapenems in Klebsiella pneumoniae isolates.

In this study, we examined the prevalence of and mechanisms of decreased susceptibility to either imipenem or meropenem in Klebsiella pneumoniae isolates. A total of 230 clinical isolates of K. pneumoniae were collected from 13 clinical laboratories from a nationwide distribution. The MICs of imipenem and meropenem were determined by the agar dilution method. To characterize the isolates with decreased susceptibility to carbapenems (MICs of >2 microg/mL), we performed polymerase chain reaction amplification of a variety of beta-lactamase genes, isoelectric focusing, and outer membrane profile analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. Three isolates (BD6, BD8, and KN16) exhibited decreased susceptibility to carbapenems with imipenem MICs of 1, 4, and 8 microg/mL and meropenem MICs of 4, 8, and 4, respectively. Isolate BD6 produced bla(TEM-1), bla(SHV-12), and bla(OXA-17); isolate BD8 produced bla(GES-3), bla(SHV-12), and bla(OXA-17); and isolate KN16 produced bla(TEM-11), bla(SHV-12), and bla(DHA-1). In all the 3 isolates, OmpK35 porin was not expressed, and in 1 isolate (KN16), OmpK36 was not expressed either. The prevalence of decreased susceptibility to carbapenems was low (1.3%), and none of them showed overt resistance to carbapenems. Decreased susceptibility to carbapenems can occur in K. pneumoniae when bla(GES-3), bla(TEM-11), bla(SHV-12), bla(OXA-17), and/or bla(DHA-1) are produced in combination with porin loss. In addition, to our knowledge, this is the 1st report of bla(OXA-17) in Enterobacteriaceae.