Xiaofei Jiang

Evolution of virulence in epidemic community-associated methicillin-resistant Staphylococcus aureus

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has recently emerged worldwide. The United States, in particular, is experiencing a serious epidemic of CA-MRSA that is almost entirely caused by an extraordinarily infectious strain named USA300. However, the molecular determinants underlying the pathogenic success of CA-MRSA are mostly unknown. To gain insight into the evolution of the exceptional potential of USA300 to cause disease, we compared the phylogeny and virulence of USA300 with that of closely related MRSA clones. We discovered that the sublineage from which USA300 evolved is characterized by a phenotype of high virulence that is clearly distinct from other MRSA strains. Namely, USA300 and its progenitor, USA500, had high virulence in animal infection models and the capacity to evade innate host defense mechanisms. Furthermore, our results indicate that increased virulence in the USA300/USA500 sublineage is attributable to differential expression of core genome-encoded virulence determinants, such as phenol-soluble modulins and α-toxin. Notably, the fact that the virulence phenotype of USA300 was already established in its progenitor indicates that acquisition of mobile genetic elements has played a limited role in the evolution of USA300 virulence and points to a possibly different role of those elements. Thus, our results highlight the importance of differential gene expression in the evolution of USA300 virulence. This finding calls for a profound revision of our notion about CA-MRSA pathogenesis at the molecular level and has important implications for design of therapeutics directed against CA-MRSA.

Complete Genome Sequence of Klebsiella pneumoniae subsp. pneumoniae HS11286, a Multidrug-Resistant Strain Isolated from Human Sputum

Klebsiella pneumoniae is an important pathogen commonly associated with opportunistic infections. Here we report the genome sequence of a strain, HS11286, isolated from human sputum in 2011 in Shanghai, China. It contains one chromosome (5.3 Mb), three multidrug resistance plasmids (∼110 kb), including a carbapenemase producer, and three small plasmids (∼3 kb).

Frequent mutations and amplifications of the PIK3CA gene in pituitary tumors

Genetic alterations in the PIK3CA gene of the phosphoinositide 3-kinase (PI3K)/AKT pathway have been found in many human tumors, but they have not been explored in pituitary tumors. We undertook the present study to explore mutations and amplifications of the PIK3CA gene in pituitary tumors. DNA sequencing and real-time quantitative PCR were used to examine mutations and amplifications respectively, on genomic DNA samples isolated from 353 cases of pituitary tumors, and immunohistostaining was used to assess PIK3CA expression. About 8 out of 91 (9%) invasive pituitary tumors versus 0 out of 262 (0%) noninvasive tumors were found to harbor somatic mutations in exons 9 and 20 of the PIK3CA gene (P<0.001), and the mutation was associated with increased disease recurrence. Genomic PIK3CA amplifications (defined as >/=4 copies) were observed in both invasive and noninvasive tumors, with a prevalence of around 20-40% in various types of pituitary tumors. PIK3CA protein overexpression was observed in cases with high PIK3CA copy number. RAS mutations were also examined and found in 6 out of the 91 (7%) invasive tumors. PIK3CA amplifications were mutually exclusive with PIK3CA or RAS mutations (P<0.001). This study demonstrated for the first time relatively common PIK3CA mutations and amplifications as well as RAS mutations and their tendency of mutual exclusivity in pituitary tumors. The data provide strong genetic evidence supporting a role of the PI3K/AKT signaling pathway in the tumorigenesis of pituitary tumors, particularly the invasive types.

Detection of Extended-Spectrum β-Lactamases in Clinical Isolates of Pseudomonas aeruginosa

ABSTRACT With the occurrence of extended-spectrum β-lactamases (ESBLs) in Pseudomonas aeruginosa being increasingly reported worldwide, there is a need for a reliable test to detect ESBLs in clinical isolates of P. aeruginosa. In our study, a total of 75 clinical isolates of P. aeruginosa were studied. Nitrocefin tests were performed to detect the β-lactamase enzyme; isoelectric focusing electrophoresis, PCR, and PCR product sequencing were designed to further characterize the contained ESBLs. Various ESBL-screening methods were designed to compare the reliabilities of detecting ESBLs in clinical isolates of P. aeruginosa whose β-lactamases were well characterized. Thirty-four of 36 multidrug-resistant P. aeruginosa clinical isolates were positive for ESBLs. blaVEB-3 was the most prevalent ESBL gene in P. aeruginosa in our study. Among the total of 34 isolates that were considered ESBL producers, 20 strains were positive using conventional combined disk tests and 10 strains were positive using a conventional double-disk synergy test (DDST) with amoxicillin-clavulanate, expanded-spectrum cephalosporins, aztreonam, and cefepime. Modifications of the combined disk test and DDST, which consisted of shorter distances between disks (20 mm instead of 30 mm) and the use of three different plates that contained cloxacillin (200 μg/ml) alone, Phe-Arg β-naphthylamide dihydrochloride (MC-207,110; 20 μg/ml) alone, and both cloxacillin (200 μg/ml) and MC-207,110 (20 μg/ml) increased the sensitivity of the tests to 78.8%, 91.18%, 85.29%, and 97.06%.

DETECTION OF EXTENDED-SPECTRUM BETA-LACTAMASES IN CLINICAL ISOLATES OF PSEUDOMONAS AERUGINOSA

ABSTRACT With the occurrence of extended-spectrum β-lactamases (ESBLs) in Pseudomonas aeruginosa being increasingly reported worldwide, there is a need for a reliable test to detect ESBLs in clinical isolates of P. aeruginosa. In our study, a total of 75 clinical isolates of P. aeruginosa were studied. Nitrocefin tests were performed to detect the β-lactamase enzyme; isoelectric focusing electrophoresis, PCR, and PCR product sequencing were designed to further characterize the contained ESBLs. Various ESBL-screening methods were designed to compare the reliabilities of detecting ESBLs in clinical isolates of P. aeruginosa whose β-lactamases were well characterized. Thirty-four of 36 multidrug-resistant P. aeruginosa clinical isolates were positive for ESBLs. blaVEB-3 was the most prevalent ESBL gene in P. aeruginosa in our study. Among the total of 34 isolates that were considered ESBL producers, 20 strains were positive using conventional combined disk tests and 10 strains were positive using a conventional double-disk synergy test (DDST) with amoxicillin-clavulanate, expanded-spectrum cephalosporins, aztreonam, and cefepime. Modifications of the combined disk test and DDST, which consisted of shorter distances between disks (20 mm instead of 30 mm) and the use of three different plates that contained cloxacillin (200 μg/ml) alone, Phe-Arg β-naphthylamide dihydrochloride (MC-207,110; 20 μg/ml) alone, and both cloxacillin (200 μg/ml) and MC-207,110 (20 μg/ml) increased the sensitivity of the tests to 78.8%, 91.18%, 85.29%, and 97.06%.

Detection of Extended-Spectrum -Lactamases in Clinical Isolates of Pseudomonas aeruginosa

ABSTRACT With the occurrence of extended-spectrum β-lactamases (ESBLs) in Pseudomonas aeruginosa being increasingly reported worldwide, there is a need for a reliable test to detect ESBLs in clinical isolates of P. aeruginosa. In our study, a total of 75 clinical isolates of P. aeruginosa were studied. Nitrocefin tests were performed to detect the β-lactamase enzyme; isoelectric focusing electrophoresis, PCR, and PCR product sequencing were designed to further characterize the contained ESBLs. Various ESBL-screening methods were designed to compare the reliabilities of detecting ESBLs in clinical isolates of P. aeruginosa whose β-lactamases were well characterized. Thirty-four of 36 multidrug-resistant P. aeruginosa clinical isolates were positive for ESBLs. blaVEB-3 was the most prevalent ESBL gene in P. aeruginosa in our study. Among the total of 34 isolates that were considered ESBL producers, 20 strains were positive using conventional combined disk tests and 10 strains were positive using a conventional double-disk synergy test (DDST) with amoxicillin-clavulanate, expanded-spectrum cephalosporins, aztreonam, and cefepime. Modifications of the combined disk test and DDST, which consisted of shorter distances between disks (20 mm instead of 30 mm) and the use of three different plates that contained cloxacillin (200 μg/ml) alone, Phe-Arg β-naphthylamide dihydrochloride (MC-207,110; 20 μg/ml) alone, and both cloxacillin (200 μg/ml) and MC-207,110 (20 μg/ml) increased the sensitivity of the tests to 78.8%, 91.18%, 85.29%, and 97.06%.

Contribution of β-Lactamases and Porin Proteins OmpK35 and OmpK36 to Carbapenem Resistance in Clinical Isolates of KPC-2-Producing Klebsiella pneumoniae

ABSTRACT Fifty-seven carbapenem-resistant Klebsiella pneumoniae isolates belonging to ST11 (50 isolates), ST423 (5 isolates), and two other sequence types were studied. All were positive for blaKPC-2, blaTEM-1, and blaCTX-M-14. SDS-PAGE analysis of six representative isolates demonstrated varied porin expression. Nevertheless, when blaKPC-2 was deleted, carbapenem resistance was markedly reduced. Additionally, SHV-12, DHA-1, and/or VIM-1 appeared to contribute to accessory carbapenemase activity. In contrast, OmpK35 and/or OmpK36 deficiency seemed to serve only as a minor cooperative factor.

First Report of a Clinical, Multidrug-Resistant Enterobacteriaceae Isolate Coharboring Fosfomycin Resistance Gene fosA3 and Carbapenemase Gene blaKPC-2 on the Same Transposon, Tn1721

ABSTRACT In order to understand the genetic background and dissemination mechanism of carbapenem resistance and fosfomycin resistance in Enterobacteriaceae isolates, we studied a clinical Escherichia coli strain HS102707 isolate and an Enterobacter aerogenes strain HS112625 isolate, both of which were resistant to carbapenem and fosfomycin and positive for the blaKPC-2 and fosA3 genes. In addition, a clinical Klebsiella pneumoniae strain HS092839 isolate which was resistant to carbapenem was also studied. A 70-kb plasmid was successfully transferred to recipient E. coli J53 by a conjugation test. PCR and Southern blot analysis showed that blaKPC-2 was located on this plasmid. The complete sequence of pHS102707 showed that this plasmid belongs to the P11 subfamily (IncP1) and has a replication gene, several plasmid-stable genes, an intact type IV secretion system gene cluster, and a composite transposon Tn1721-Tn3 that harbored blaKPC-2. Interestingly, a composite IS26 transposon carrying fosA3 was inserted in the Tn1721-tnpA gene in pHS102707 and pHS112625, leading to the disruption of Tn1721-tnpA and the deletion of Tn1721-tnpR. However, only IS26 with a truncated Tn21-tnpR was inserted in pHS092839 at the same position. To our knowledge, this is the first report of fosA3 and blaKPC-2 colocated in the same Tn1721-Tn3–like composite transposon on a novel IncP group plasmid.

Characterization of the genetic environment of the blaKPC-2 gene among Klebsiella pneumoniae isolates from a Chinese Hospital.

Infection caused by carbapenem-resistant Klebsiella pneumoniae has become a major healthcare threat and KPC-2 enzyme is a dominant factor mediating carbapenems resistance in K. pneumoniae. This study was designed to determine the genetic environment of blaKPC-2, which prevailed in clinical K. pneumoniae isolates recovered in Huashan Hospital, Shanghai, China. Forty-two clinical isolates were included in this study by blaKPC-2 screening. After multilocus sequence typing and plasmid analyses of PCR-based replicon typing (PBRT), junction PCR, mapping PCR and crossing PCR assays, primer walking, and amplicon sequencing were used to analyze the genetic environment of the blaKPC-2 gene. ST423, ST65, ST977, and ST11 were all detected in KPC-2-producing K. pneumoniae. Two types of blaKPC-2-bearing genetic structure were found: Tn1721-blaKPC-2-Tn3 and Tn1721-blaKPC-2-ΔTn3-IS26; and were carried in IncX and IncFII plasmids, respectively. In conclusion, the genetic environment of the blaKPC-2 gene was diverse and Tn1721-blaKPC-2-ΔTn3-IS26 was dominant in clinical K. pneumoniae isolates in Huashan Hospital. This study sheds some light on the genetic environment and should foster further studies about the mechanism of the blaKPC-2 dissemination.

Transcription of integron-harboured gene cassette impacts integration efficiency in class 1 integron

Class 1 integrons play important roles in the dissemination of antibiotic resistance genes among bacteria. Generally, class 1 integron consists of an integrase gene (intI1), a recombination site (attI1) and a promoter (Pc) that drives the transcription of the downstreamed gene cassettes. Occasionally, there is a second promoter P2 downstream of the Pc promoter. Several Pc variants and Pc–P2 combinations have been defined and they display different transcription strengths, but the influence of the transcription of integron‐harboured gene cassette on the integration efficiency has never been comprehensively studied. In this study, the integration frequencies of gene cassettes into the attI1 sites that downstream of four different Pc variants as well as their combinations with P2 promoter were measured. The results showed that there was an inverse correlation between the strength of Pc promoter and the integration efficiency and, with the same Pc promoter, the integration efficiency was significantly decreased when a P2 promoter preceded the attI1 site. Our findings indicate there is a relationship between the transcription of integron‐harboured gene cassette and the integration of exogenous gene cassettes. The interrelationship between these two relatively independent processes may throw a light on our understanding the regulation system of class 1 integron.