Liyan Ye

Molecular epidemiology and virulence factors of pyogenic liver abscess causing Klebsiella pneumoniae in China

The molecular epidemiology and prevalence of virulence factors of isolates from patients with Klebsiella pneumoniae liver abscess (KLA) in mainland China are unknown. Klebsiella pneumoniae isolates were obtained from drainage samples aseptically collected from patients with pyogenic liver abscess (PLA). The genetic similarity of KLA isolates was analyzed by pulsed-field gel electrophoresis. The hypermucoviscosity (HV) phenotype was identified by a positive string test. The K1 and K2 genotypes, the pLVPK-derived genetic loci, aerobactin gene, kfu and alls were detected by PCR amplification. The sequence types (STs) were identified by multilocus sequence typing. Among the 51 non-repetitive KLA isolates, 49 PFGE types have been identified. In total, 19 (37.2%) and 14 (27.4%) of the 51 KLA isolates belonged to clonal complex (CC) 23 and CC65, respectively, while the other 18 isolates (35.3%) were defined as other STs. CC23 consisted of only K1 strains, while CC65 included only K2 strains. All non-K1/K2 strains were classified as STs other than CC23 and CC65. Approximately 70.6% (36/51) of KLA isolates exhibited an HV phenotype. Both K1 and K2 isolates presented significantly higher prevalence of the pLVPK-derived loci than non-K1/K2 isolates. The K1 isolates had a significantly higher prevalence of the kfu and allS genes than K2 and non-K1/K2 isolates, while the K2 isolates exhibited higher repA prevalence than K1 and non-K1/K2 isolates. The majority of KLA isolates belonged to CC23K1 and CC65K2, while other STs with non-K1/K2 capsular types have also been identified. The virulent factors exhibited diverse distribution among the different clones of KLA isolates.

Similarity and Divergence of Phylogenies, Antimicrobial Susceptibilities, and Virulence Factor Profiles of Escherichia coli Isolates Causing Recurrent Urinary Tract Infections That Persist or Result from Reinfection

ABSTRACT In order to obtain a better molecular understanding of recurrent urinary tract infection (RUTI), we collected 75 cases with repeatedly occurring uncomplicated UTI. The genetic relationships among uropathogenic Escherichia coli (UPEC) isolates were analyzed by pulsed-field gel electrophoresis. While 39 (52%) of the RUTI cases were defined as “persistence” of the same strain as the primary infecting strain, 36 (48%) were characterized by “reinfection” with a new strain that is different from the primary strain. We then examined the antimicrobial susceptibilities and phylogenetic backgrounds of 39 persistence and 86 reinfection UPEC isolates, and screened 44 virulence factor (VF) genes. We found that isolates had significant differences in the following: placement in phylogenetic group B2 (41% versus 21%; P = 0.0193) and the presence of adhesin genes iha (49% versus 28%; P = 0.0233) and papG allele I′ (51% versus 24%; P = 0.003), iron uptake genes fyuA (85% versus 58%; P = 0.0037), irp-2 (87% versus 65%; P = 0.0109), and iutA (87% versus 58%; P = 0.0014), and an aggregate VF score (median, 11 versus 9; P = 0.0030). In addition, 41% of persistence strains harbored three adhesin genes simultaneously, whereas 22% of reinfection isolates did (P = 0.0289). Moreover, 59% versus 29% (P = 0.0014) of persistence and reinfection isolates contained seven types of iron uptake genes. Taken together, the antimicrobial susceptibilities of UPEC isolates had little effect on the RUTI. Compared with reinfection strains, persistence UPEC isolates exhibited higher VF scores and carried more VF genes than may be involved in the development and progression of RUTI.

Robinsoniella peoriensis Bacteremia in a Patient with Pancreatic Cancer

ABSTRACT Robinsoniella peoriensis was recently identified as a Gram-positive, spore-forming, anaerobic rod originally isolated from swine manure storage pits. We describe here a case of R. peoriensis bacteremia in a 42-year-old male with pancreatic cancer. The identification was confirmed by unique phenotypic profiles and partial 16S rRNA gene sequences.

Comparative study of MALDI-TOF MS and VITEK 2 in bacteria identification

BACKGROUND Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been introduced in diagnostic microbiology laboratories for the identification of bacterial and yeast strains isolated from clinical samples. This study aimed to evaluate the accuracy of MALDI-TOF MS in clinical microbiology diagnosis by comparing it with commonly-used VITEK 2 or gene sequencing. METHODS The performances of MALDI-TOF MS and VITEK 2 were compared retrospectively for identifying routine isolates. Discrepancies were analyzed by gene sequencing analysis of the 16S genes. RESULTS For 1,025 isolates, classified as 55 species of 25 genera, 1,021 (99.60%) isolates were accurately identified at the genus level, and 957 (93.37%) isolates at the species level by using MALDI-TOF MS. A total of 949 (92.59%) isolates were completely matched by both methods. Both methods found 76 unmatched isolates among which one strain had no definite identification by MALDI-TOF MS and VITEK 2 respectively. However, MALDI-TOF MS made no errors at the genus level while VITEK 2 made 6 (0.58%) errors at the genus level. At the species level, the identification error rates for MALDI-TOF MS and VITEK 2 were 5.56% and 6.24%, respectively. CONCLUSIONS With a lower identification error rate, MALDI-TOF MS has better performance than VITEK 2 in identifying bacteria found routinely in the clinical laboratory. It is a quick and cost-effective technique, and has the potential to replace conventional phenotype methods in identifying common bacterial isolates in clinical microbiology laboratories.

Characterization of Clinical Multidrug-Resistant Escherichia coli and Klebsiella pneumoniae Isolates, 2007–2009, China

Various resistance mechanisms facilitate the emergence and spread of multidrug-resistance (MDR) phenotypes of Escherichia coli and Klebsiella pneumoniae. To elucidate the MDR mechanisms of E. coli and K. pneumoniae in China, we analyzed the antimicrobial susceptibilities of strains isolated from clinical samples in a large tertiary care hospital in Beijing, China, during 2007-2009 and characterized the isolates with a cefotaxime-ciprofloxacin-amikacin (CTX-CIP-AK) resistance pattern. In total, 98 and 52 clinical isolates of E. coli and K. pneumoniae, respectively, with a CTX-CIP-AK resistance pattern were subjected to antimicrobial susceptibility testing and screening of common β-lactamase genes, plasmid-mediated quinolone resistance (PMQR) genes, quinolone resistance-determining region (QRDR) substitutions, and 16S rRNA methylase genes by polymerase chain reaction amplification and DNA sequencing. Pulsed-field gel electrophoresis (PFGE) was used to determine the genetic relatedness of the isolates. Approximately 6.86% and 8.05% of the clinical E. coli and K. pneumoniae isolates, respectively, exhibited MDR phenotypes. The MDR K. pneumoniae isolates exhibited significantly higher ceftazidime resistance than the MDR E. coli isolates (90.4% vs. 76.5%, p=0.0339); a similar result was noted for piperacillin-tazobactam resistance (28.8% vs. 2%, p=0.0001). The common resistance determinants among the MDR E. coli and K. pneumoniae isolates were as follows: CTX-M (88.8% vs. 82.7%), PMQR genes (70.4% vs. 90.4%), gyrA mutations (100% vs. 90.4%), and 16S rRNA methylase genes (93.9% vs. 94.2%). Half (50%) of the MDR E. coli isolates belonged to phylogenetic group D, followed by group A (39.8%). For the E. coli isolates, 94 PFGE patterns and 23 clusters were identified, whereas 51 PFGE patterns and 11 clusters were identified for the K. pneumoniae isolates. Clinical E. coli and K. pneumoniae isolates seem to have a low prevalence of MDR phenotypes in China. The great genetic variation indicates a considerable transmission of common resistance determinants, including a high prevalence of QRDR substitutions in E. coli and K. pneumoniae.

Nosocomial Outbreak of OXA-48-Producing Klebsiella pneumoniae in a Chinese Hospital: Clonal Transmission of ST147 and ST383.

Background In China, the spread and outbreak of OXA-48-producing Enterobacteriaceae remains largely unknown. Methods OXA-48-producing isolates were analyzed for genetic relatedness by pulsed-field gel electrophoresis (PFGE), antimicrobial susceptibility by E-test, and sequence type (ST) by multilocus sequence typing. S1-PFGE and southern blotting were used for plasmid profiling, and PCR and subsequent sequencing were performed to determine the genetic environment of blaOXA-48 gene. Results In total, 37 non-duplicated OXA-48-producing K. pneumoniae (OXAKp) isolates were recovered. From December 2013 to August 2014, an outbreak was observed at a respiratory ICU. The 37 isolates of K. pneumoniae were categorized into four PFGE types (A, B, C, and D). The predominant strains associated with the outbreak were strains with PFGE type A and B, which belonged to ST383 and ST147, respectively. Plasmid sequencing revealed that the blaOXA-48-carrying plasmid is 69,069 bp in length and belongs to the IncL/M incompatibility group. Sequence analysis revealed that the IS1999 element was located upstream of the blaOXA-48 gene and was truncated by IS1R. Conclusions In this study, the dissemination and outbreak of OXAKp isolates were clonal, and ST147 and ST383 K. pneumoniae were the predominant clones that were associated with the outbreak. Meanwhile, the horizontal transfer of plasmids potentially mediate the spread of blaOXA-48 gene between different K. pneumoniae strains.

Predictive value of procalcitonin for excluding bloodstream infection: Results of a retrospective study and utility of a rapid, quantitative test for procalcitonin

Objectives To assess retrospectively the diagnostic value of procalcitonin (PCT) in excluding suspected bloodstream infection, establish cut-off values for PCT levels, and compare PCT with other clinical markers. Methods The predictive accuracy of different continuous parameters was estimated by univariate analysis of the area under the receiver operating characteristic curve. Optimized cut-off points for the parameters were selected according to the maximum Youden index values, which in turn were used to define positive and negative predictive values of different parameters in diagnosing bloodstream infection. Results The PCT level yielded a statistically significant area under the receiver operating characteristic curve of 0.765, with a best cut-off value of 0.80 ng/ml (83% sensitivity; 65% specificity, Youden index, J = 0.48). Positive and negative predictive values at this cut-off value were 38% and 94%, respectively. Mann–Whitney U-test revealed significantly higher values for PCT, C-reactive protein and percentage of neutrophils, but not for white blood cell count, in patients with bloodstream infection. Conclusions The serum PCT level can potentially be used as surrogate marker to exclude bacteraemia and to inform critical management decisions regarding antibiotic usage, in patients admitted with suspected bloodstream infection.

Clinical and microbiological characterization of Staphylococcus lugdunensis isolates obtained from clinical specimens in a hospital in China.

BackgroundSeveral reports have associated Staphylococcus lugdunensis with the incidence of severe infection in humans; however, the frequency and prevalence of this microorganism and thus the propensity of its antimicrobial drug resistance is unknown in China. The objective of the current study was to determine the prevalence of Staphylococcus lugdunensis among six hundred and seventy non-replicate coagulase negative Staphylococcus (CoNS) isolates collected in a 12-month period from clinical specimens in the General Hospital of the People’s Liberation Army in Beijing, China.ResultsFive (0.7%) of the 670 isolates of CoNS were identified as S. lugdunensis. Whereas three isolates were resistant to erythromycin, clindamycin, and penicillin and carried the ermC gene and a fourth one was resistant to cefoxitin and penicillin and carried the mecA gene, one isolate was not resistant to any of the tested antimicrobials. Pulse field gel electrophoretic analysis did not reveal widespread epidemiological diversity of the different isolates.ConclusionHence, even though S. lugdunensis may be yet unrecognized and undefined in China, it still might be the infrequent cause of infection and profound multi-drug resistance in the same population.

Performance of the VITEK MS matrix-assisted laser desorption ionization-time of flight mass spectrometry system for rapid bacterial identification in two diagnostic centres in China

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) MS systems was not officially launched for diagnostic use in clinical microbiology laboratories in China until 2012. Here, we report the findings from the first large-scale evaluation study of VITEK MS for routine bacterial identification in two major diagnostic centres in Beijing and Hong Kong. A total of 2266 unique isolates representing 56 genera and 127 species were analysed, and results were compared to those obtained by VITEK 2. Any discrepancies were resolved by 16S rRNA sequencing. Overall, VITEK MS provided correct identification for 2246 (99.1%) isolates, including 2193 (96.8 %) with correct species-level identifications and 53 (2.3 %) matched at the genus level only. VITEK MS surpassed VITEK 2 consistently in species-level identification of important pathogens, including non-Enterobacteriaceae Gram-negative bacilli (94.7 versus 92 %), staphylococci (99.7 versus 92.4 %), streptococci (92.6 versus 79.4 %), enterococci (98.8 versus 92.6 %) and Clostridium spp. (97.3 versus 55.5 %). The findings demonstrated that VITEK MS is highly accurate and reliable for routine bacterial identification in clinical settings in China.

Prevalence and Antimicrobial Susceptibility of Hypervirulent Klebsiella pneumoniae Isolates in China

TO THE EDITOR—Recently, Li et al reported that an increasing proportion of hypervirulent Klebsiella pneumoniae (hvKP) among clinical K. pneumoniae isolates has been identified in China and that the hvKP strains exhibited an increasing antimicrobial resistance [1]. Our concern is that their study may have significant impact on the empiric anti-infection treatment of serious infections caused by hvKP. Therefore, we performed this study to clarify the hypermucoviscosity (HV) and antimicrobial susceptibility of clinical K. pneumoniae isolates causing pyogenic liver abscess (PLA) and metastatic infectious disease (MID). In contrast to the findings from Li et al [1], we did not find an increasing prevalence of HV and antimicrobial resistance among those isolates. Thus, there are several issues of concern that need to be discussed. First, the genetic relatedness of clinical isolates should be further identified by molecular analysis. Li et al stated that “cases of MID were diagnosed in patients with K. pneumoniae–positive culture obtained from 1 or more sites in addition to a clear infection site, even if the abscess fluid culture was negative for K. pneumoniae” [1]. We analyzed 67 patients who suffered from blood and other infections simultaneously. Comparisons of their pulsed-field gel electrophoresis profiles revealed that 16.4% (11/67) of the patients were infected by different clones. Therefore, it is unreliable to identify MID-causing isolates just by isolating K. pneumoniae from different sites, not to mention the negative cultures for some abscesses. Second, in the study reported by Li et al, the K. pneumoniae strains with a positive string test were designated as hvKP [1]. In this study, only 70.6% (35 of 51) of PLAcausing and 32.1% (18 of 56) of MIDcausing K. pneumoniae isolates exhibited HV. Because the factors other than HV are required for the systemic dissemination of K. pneumoniae [2], and because whether all hvKP are hypermucoviscous remains unclear [3], hvKP should be defined by genomic background, rather than by HV [4, 5]. Therefore, defining hvKP only by HV likely leads to a biased result. For example, contrary to the findings from Li et al [1], we found that the PLA-causing K. pneumoniae isolates exhibited an absolute susceptibility to the commonly used antibiotics. Finally, Li et al conducted their study with only 88 patients from a single hospital in 3 years, and the average annual number of case patients is <30 [1]. Therefore, the sample size of that study cannot be considered to be the true reality even with a large sample size. From a statistical point of view, it is not suitable for an annual proportion analysis of hvKP. Meanwhile, on the epidemiological aspect, it is very unreliable to conclude that “hvKP strains are being isolated from patients in China” just by a single-center study with a small sample size in a special hospital. In conclusion, a truly objective and scientific conclusion should be obtained from a multicenter study with a large number of samples collected from patients in the representative regions/hospitals nationally.