Prevalence of ST1193 clone and IncI1/ST16 plasmid in E-coli isolates carrying blaCTX-M-55 gene from urinary tract infections patients in China.

To study molecular epidemiology of CTX-M-55-carrying Escherichia coli isolates from urinary tract infections (UTIs) in China. 111 blaCTX-M-55-positive E.coli isolates from UTIs patients in China were studied. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to analyze the homologies among the strains. Conjugation experiments, S1nuclease PFGE and PCR analysis were performed to characterize plasmids harboring blaCTX-M-55 and their genetic environment. 111 isolates were clustered into 86 individual pulsotypes and three clusters by PFGE. Fifty-five (49.5%) of the isolates belonged to 8 STs. Most of the ST1193 isolates belonged to one PFGE cluster. Transconjugants (n = 45) derived from randomly selected blaCTX-M-55 donors (n = 58), were found to contain a single 90-kb conjugative plasmid, which mainly belonged to the IncI1 groups (34, 76%). Among the IncI1 plasmids, the blaCTX-M-55/IncI1/ST16 predominated (23/34, 68%). The blaTEM-1 and aac (3')-II genes were frequently detected on the IncI1 plasmids, and the insertion of ISEcp1 or IS26 was observed at the 48 bp or 45 bp upstream of the start codon of blaCTX-M-55 gene. The dissemination of blaCTX-M-55 gene among E. coli UTI isolates, appeared to be due to both the major clonal lineage of ST1193 and the horizontal transfer of epidemic plasmid IncI1/ST16.

Direct detection of methicillin-resistant Staphylococcus aureus in sputum specimens from patients with hospital-associated pneumonia using a novel multilocus PCR assay.

Methicillin-resistant Staphylococcus aureus (MRSA) is a significant cause of hospital-associated pneumonia (HAP). The rapid identification of MRSA would be beneficial for early diagnosis. The study aimed to evaluate a multilocus, fluorescence-based PCR assay based on the detection of mecA and nuc genes for identification of S. aureusin lower respiratory tract (LRT) specimens. Sensitivity and specificity of the PCR assay were analyzed. Clinical evaluation for the assay was performed using LRT specimens from patients with HAP, and the sensitivity, specificity, positive and negative predictive values (PPV and NPV) were evaluated in comparison with semi-quantitative culture methods. The result showed the assay provided positive identification of all MRSA reference strains with a limit of detection for MRSA of 4 × 103 CFU/mL. Compared with semi-quantitative culture, the sensitivity, specificity, PPV and NPV were 100%, 89.6%, 75.0%, and 100%, respectively. A positive correlation between MRSA bacterial colonies and PCR copy number was found. The specificity and PPV reached 96.6% and 89.7% respectively, if the PCR copy number reached a definite positive threshold of 5.96 × 105. It suggested that this novel multilocus, fluorescence-based PCR assay proved to be a fast, sensitive and specific tool for direct detection of MRSA from LRT specimens.

The impact of spgM, rpfF, rmlA gene distribution on biofilm formation in Stenotrophomonas maltophilia.

BACKGROUND: Stenotrophomonas maltophilia is emerging as one of the most frequently found bacteria in chronic pulmonary infection. Biofilm is increasingly recognized as a contributing factor to disease pathogenesis. In the present study, a total of 37 isolates of S. maltophilia obtained from chronic pulmonary infection patients were evaluated to the relationship between biofilm production and the relative genes expression. METHODS: The clonal relatedness of isolates was determined by pulse-field gel electrophoresis. Biofilm formation assays were performed by crystal violet assay, and confirmed by Electron microscopy analysis and CLSM analysis. PCR was employed to learn gene distribution and expression. RESULTS: Twenty-four pulsotypes were designated for 37 S. maltophilia isolates, and these 24 pulsotypes exhibited various levels of biofilm production, 8 strong biofilm-producing S. maltophilia strains with OD492 value above 0.6, 14 middle biofilm-producing strains with OD492 average value of 0.4 and 2 weak biofilm-producing strains with OD492 average value of 0.19. CLSM analysis showed that the isolates from the early stage of chronic infection enable to form more highly structured and multilayered biofim than those in the late stage. The prevalence of spgM, rmlA, and rpfF genes was 83.3%, 87.5%, and 50.0% in 24 S. maltophilia strains, respectively, and the presence of rmlA, spgM or rpfF had a close relationship with biofilm formation but did not significantly affect the mean amount of biofilm. Significant mutations of spgM and rmlA were found in both strong and weak biofilm-producing strains. CONCLUSION: Mutations in spgM and rmlA may be relevant to biofilm formation in the clinical isolates of S. maltophilia.

Glycopeptide minimum inhibitory concentration creep among meticillin-resistant Staphylococcus aureus from 2006-2011 in China.

Vancomycin minimum inhibitory concentration (MIC) creep has recently been demonstrated by many countries but is rarely reported in China. In this study, a total of 1411 meticillin-resistant Staphylococcus aureus (MRSA) isolates were collected from six hospitals in China during the period 2006-2011 and the MICs of vancomycin, teicoplanin and linezolid were determined by broth microdilution according to Clinical and Laboratory Standards Institute (CLSI) guidelines. MIC50 and MIC90 values (MICs required to inhibit the growth of 50% and 90% of organisms, respectively) as well as geometric mean (GM) MICs were calculated for all isolates in each year, and MIC creep for the drugs was evaluated. All of the MRSA isolates were susceptible to vancomycin and linezolid. Overall, the vancomycin GM MIC of MRSA isolates was 0.906, 0.952, 0.956, 0.947, 1.013 and 1.040 mg/L, with a significantly increasing trend over the years (P<0.001). Percentages of MRSA isolates with a vancomycin MIC above 1 µg/mL (2 µg/mL≥MIC>1 µg/mL) were 26.0%, 23.5%, 21.6%, 27.8%, 30.6% and 42.8% from 2006-2011, respectively, and increased over time (P<0.005). The teicoplanin GM MIC increased rapidly from 0.749 mg/L in 2008 to 0.973 mg/L in 2011, and ca. 5% of isolates were resistant to teicoplanin in the period according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. MIC shifts were not found for linezolid (P>0.05). In conclusion, a tendency towards decreasing susceptibility to glycopeptides in MRSA has emerged in China.

[Clinical significance of virulence-related genes of type III secretion system of Pseudomonas aeruginosa].

OBJECTIVE: To study the clinical significance of virulence genes exo U and exo S of type III secretion system (TTSS) of Pseudomonas aeruginosa (PA). METHODS: One hundred and eighty-nine clinical isolates of PA were collected from five hospitals. The incidence of virulence genes exo U and exo S in PA were determined with PCR. Minimum inhibitory concentration of anti-bacterial drug for PA was determined with microdilution method. The clinical features and outcomes of 60 hospitalized patients colonized or infected with exo U+/exo S- positive or exo U-/exo S+ positive PA isolated from sputum were analyzed retrospectively. Data were processed with chi-square test. RESULTS: Among the 189 PA isolates, 85.2% (161/189) harbored TTSS genes, including exo U-/exo S+ type (120 isolates), exo U+/exo S- type (31 isolates), exo U-/exo S- type (7 isolates), and exo U+/exo S+ type (3 isolates). 72.0% (72/100) isolates from sputum and 81.5% (44/54) isolates from blood belonged to exo U-/exo S+ genotype. Compared with those of TTSS-negative isolates, the antimicrobial resistance of TTSS-positive isolates to cefoperazone/sulbactam, ceftazidime, amikacin, and cefepime were lower (with χ² value respectively 10.1, 16.1, 9.3, 33.8, P values all below 0.01). The antimicrobial resistance to all examined drug between exo U-/exo S+ type and exo U+/exo S- type isolates was close (with χ² values from 0.08 to 2.04, P values all above 0.05). Patients detected with exo U+/exo S- positive PA isolated from sputum were significantly associated with PA infection, and they usually had history of tracheal intubation, ICU hospitalization, and combined use of drugs for anti-infection treatment. Patients detected with exo U-/exo S+ positive PA isolated from sputum were significantly associated with PA colonization, which had basic lung disease and better outcome than the former infection type. CONCLUSIONS: The TTSS exists in most clinical isolates of PA. Detection of exo U or exo S of PA isolated from sputum is helpful for the analysis of clinical features and outcome of patients.

[Research of differential expression of virulence-related proteins of T3SS in Pseudomonas aeruginosa].

OBJECTIVE: To analyze the difference of virulence-related protein concerned with type III secretion system (T3SS) in Pseudomonas aeruginosa during the changes of antibiotic sensitivity and interpret the clinical patient data to explore the relationship between the changes in resistance and variance of virulence. METHODS: The isolates of Pseudomonas aeruginosa was isolated from the respiratory tract of a same patient with an altered sensitivity of antibiotics. It turned out to be one clone. The homolog of isolates was determined by ERIC-PCR. The Kirby-Bauer antibiotic testing was employed to detect the sensitivity of antibiotics of isolates. PCR was used to detect the gene of T3SS and virulence of isolates and two-dimensional gel electrophoresis to compare the whole-cell proteins. The mass spectrometry was employed to analyze a variety of protein spots. The relevant information was retrieved from protein databases. Clinical record was collected to study the relationship of clinical features, bacteria resistance and virulence-associated protein. RESULTS: One subject was diagnosed with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) complicated with pneumonia. Pseudomonas aeruginosa was isolated from his respiratory tract three times in one year. Sensitivity spectrum of isolates were as follows: sensitivity, multi-drug resistant (MDR) and pan-drug resistance (PDR). Virulence gene was exo U+/exo S-. Twenty-one differentially expressed proteins were revealed by two-dimensional gel electrophoresis in three isolates. The functions of 11 proteins were definite. Only 9 proteins were associated with basal bacterial metabolism. Disulfide oxidoreductase A (DsbA) corresponded to the variation of virulence and sensitivity spectrum. Clinical record revealed that the severe lung infection was caused by the PDR strain and the patient died within one month. CONCLUSIONS: The sensitivity spectrum and virulence of Pseudomonas aeruginosa may undergo changes when there is an alteration of eco-environment during colonization and infection over a long period of time. DsbA plays a key role in the variety of virulence.

[In vitro antimicrobial activity of piperacillin-sulbactam and other 7 antimicrobial agents against non-fermentative bacilli].

OBJECTIVE: To evaluate the in vitro antimicrobial activity of piperacillin-sulbactam against clinical isolates of non-fermentative bacilli isolated from common infections. METHODS: Microdilution was employed to study the antimicrobial resistance. RESULTS: A total of 770 strains were collected from 6 hospitals in Guangzhou, including Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, Burkholderia cepacia, Flavobacterium, and Alcaligenes. Compared with other ß-lactams, piperacillin-sulbactam displayed the highest activity against all the isolates of P.aeruginosa, especially for imipenem non-sensitive isolates, with the susceptibility of 71.9% and 55.8%, respectively. Piperacillin-sulbactam and cefoperazone-sulbactam kept good activity against imipenem sensitive isolates of A.baumannii, with the susceptibility of 71.0% and 73.0%, respectively. For the strains of Burkholderia cepacia, 69% strains exhibited minimal inhibitory concentration (MIC) of ≤ 16 mg/L for piperacillin-sulbactam. For the strains of Flavobacterium, and Alcaligenes, piperacillin-sulbactam also had excellent activity, with the susceptibility of 70.2% and 94.4%, respectively. CONCLUSION: Piperacillin-sulbactam exhibits good activity again non-fermentative bacilli, especially for imipenem non-sensitive isolates of P.aeruginosa.