Fabrication of levofloxacin-loaded porcine acellular dermal matrix hydrogel and functional assessment in urinary tract infection.

Bacterial cystitis, a commonly occurring urinary tract infection (UTI), is renowned for its extensive prevalence and tendency to recur. Despite the extensive utilization of levofloxacin as a conventional therapeutic approach for bacterial cystitis, its effectiveness is impeded by adverse toxic effects, drug resistance concerns, and its influence on the gut microbiota. This study introduces Lev@PADM, a hydrogel with antibacterial properties that demonstrates efficacy in the treatment of bacterial cystitis. Lev@PADM is produced by combining levofloxacin with decellularized porcine acellular dermal matrix hydrogel and exhibits remarkable biocompatibility. Lev@PADM demonstrates excellent stability as a hydrogel at body temperature, enabling direct administration to the site of infection through intravesical injection. This localized delivery route circumvents the systemic circulation of levofloxacin, resulting in a swift and substantial elevation of the antimicrobial agent's concentration specifically at the site of infection. The in vivo experimental findings provide evidence that Lev@PADM effectively prolongs the duration of levofloxacin's action, impedes the retention and invasion of E.coli in the urinary tract, diminishes the infiltration of innate immune cells into infected tissues, and simultaneously preserves the composition of the intestinal microbiota. These results indicate that, in comparison to the exclusive administration of levofloxacin, Lev@PADM offers notable benefits in terms of preserving the integrity of the bladder epithelial barrier and suppressing the recurrence of urinary tract infections.

Effect of tocilizumab plus corticosteroid on clinical outcome in patients hospitalized with severe fever with thrombocytopenia syndrome: A randomized clinical trial.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging viral hemorrhagic fever with high fatality rates. The blockade of pro-inflammatory cytokines presents a promising therapeutic strategy. METHODS: We conducted a randomized clinical trial at the 154th hospital, Xinyang, Henan Province. Eligible patients with severe SFTS disease were randomly assigned in a 1:2 ratio to receive either a single intravenous infusion of tocilizumab plus usual care; or usual care only. The primary outcome was the clinical status of death/survival at day 14, while secondary outcomes included improvement from baseline in liver and kidney damage and time required for hospital discharge. The efficacy of tocilizumab plus corticosteroid was compared to those receiving corticosteroid alone. The trial is registered with the Chinese Clinical Trial Registry website (ChiCTR2300076317). RESULTS: 63 eligible patients were assigned to the tocilizumab group and 126 to the control group. The addition of tocilizumab to usual care was associated with a reduced death rate (9.5%) compared to those received only usual care (23.0%), with an adjusted hazard ratio (aHR) of 0.37 (95% confidence interval [CI], 0.15 to 0.91, P = 0.029). Combination therapy of tocilizumab and corticosteroids was associated with a significantly reduced fatality (aHR, 0.21; 95% CI, 0.08 to 0.56; P = 0.002) compared to those receiving corticosteroids alone. CONCLUSIONS: A significant benefit of reducing fatality in severe SFTS patients was observed by using tocilizumab. A combined therapy of tocilizumab plus corticosteroids was recommended for the therapy of severe SFTS.

Plasmid-mediated quinolone resistance in extended-spectrum-ß-lactamase- and AmpC ß-lactamase-producing Serratia marcescens in China.

We investigated the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants and examined the association of these determinants with extended-spectrum ß-lactamases (ESBLs) and/or plasmid-mediated AmpC ß-lactamases (pAmpCs) in Serratia marcescens isolates in China. In this study, the presence of PMQR determinants was significantly related to the coproduction of ESBLs and/or pAmpCs (CTX-M-14, SHV-5, DHA-1, and ACT-1), among which CTX-M-14 was the most common gene type.

Testing the mutant selection window in rabbits infected with methicillin-resistant Staphylococcus aureus exposed to vancomycin.

OBJECTIVES: To test the mutant selection window (MSW) hypothesis with Staphylococcus aureus exposed to vancomycin in an animal model and to compare in vivo and in vitro exposures that restrict the enrichment of resistant mutants. METHODS: Local infection with S. aureus was established in rabbits, and the infected animals were treated with various doses of twice-daily vancomycin (half-life 6 h) for 3 consecutive days to provide antibiotic concentrations below the MIC, between the MIC and the mutant prevention concentration (MPC), and above the MPC. Changes in susceptibility and the numbers of surviving organisms were monitored daily on agar plates containing 2× and 4× MIC of vancomycin. RESULTS: S. aureus lost vancomycin susceptibility when drug concentrations at the site of infection fluctuated between the lower and upper boundaries of the MSW, defined in vitro as the MIC(99) and the MPC, respectively. Both boundaries were determined in vitro, before starting animal studies. The value at which resistant mutants are not enriched in vivo was estimated as an AUC(24)/MPC value of ∼15 h, where AUC(24) is the area under the drug concentration time curve in a 24 h interval. The estimated anti-mutant AUC/MIC ratio in vivo was ≥200 h. CONCLUSIONS: These findings support the MSW hypothesis and the anti-mutant AUC/MIC ratio estimated in vivo is consistent with that reported in in vitro studies. Keeping vancomycin concentrations above the MPC or AUC(24)/MPC >15 h is a straightforward way to restrict the acquisition of resistance.

Emergence of serogroup C meningococcal disease associated with a high mortality rate in Hefei, China.

BACKGROUND: Neisseria meningitidis serogroup C has emerged as a cause of epidemic disease in Hefei. The establishment of serogroup C as the predominant cause of endemic disease has not been described. METHODS: We conducted national laboratory-based surveillance for invasive meningococcal disease during 2000-2010. Isolates were characterized by pulsed-field gel electrophoresis and multilocus sequence typing. RESULTS: A total of 845 cases of invasive meningococcal disease were reported. The incidence increased from 1.25 cases per 100,000 population in 2000 to 3.14 cases per 100,000 in 2003 (p < 0.001), and peaked at 8.43 cases per 100,000 in 2005. The increase was mainly the result of an increase in the incidence of serogroup C disease. Serogroup C disease increased from 2/23 (9%) meningococcal cases and 0.11 cases per 100,000 in 2000 to 33/58 (57%) cases and 1.76 cases per 100,000 in 2003 (p < 0.01). Patients infected with serogroup C had serious complications more frequently than those infected with other serogroups. Specifically, 161/493 (32.7%) cases infected with serogroup C had at least one complication. The case-fatality rate of serogroup C meningitis was 11.4%, significantly higher than for serogroup A meningitis (5.3%, p = 0.021). Among patients with meningococcal disease, factors associated with death in univariate analysis were age of 15-24 years, infection with serogroup C, and meningococcemia. CONCLUSIONS: The incidence of meningococcal disease has substantially increased and serogroup C has become endemic in Hefei. The serogroup C strain has caused more severe disease than the previously predominant serogroup A strain.

Surveillance of antimicrobial susceptibility patterns among Stenotrophomonas maltophilia isolated in China during the 10-year period of 2005-2014.

The antimicrobial resistance pattern was studied among 300 Stenotrophomonas maltophilia isolates collected from 300 nonduplicated inpatients. The average age of the patients was 73.5 ± 14.6 years old. Isolates nonsusceptible to levofloxacin, chloramphenicol, minocycline or trimethoprim/sulfamethoxazole (SXT) were more prevalent in the 99 patients aged over 81 years than in the other patients (p < 0.05). Multidrug resistance was found to be significantly associated with isolates recovered from the blood (72.7%, p = 0.000) and elderly patients (31.3%, p = 0.003). A trend of increased resistance to antimicrobials and higher and higher frequencies of multidrug-resistant isolates were observed through the 10-year period, wherein the percentage of isolates resistant to SXT was significantly changed from 29.7% in 2005-2009 to 47.1% in 2010-2014 (p = 0.02). Clinicians are recommended to pay special attention to the possibility of multidrug-resistant S. maltophilia infection in elderly inpatients.

The Regulation of Seventeen Inflammatory Mediators are Associated with Patient Outcomes in Severe Fever with Thrombocytopenia Syndrome.

Severe fever with thrombocytopenia syndrome (SFTS) as an emerging infection disease results in high morbidity and mortality in China. In this study, the circulating levels of 36 inflammatory mediators in 33 SFTS patients on days 3-7, 8-12 and 13-20 post-illness were measured by a multiplex Luminex® system dynamically. Among the patients, 15 severe patients recovered, 11 severe patients died within three weeks. We found IL-1RA, IL-6, IL-15, IL-10, TNF-α, IFN-γ, G-CSF, eotaxin, IL-8, IP-10, MCP-1, MIP-1α, MIP-1ß and fractalkine were significantly upregulated in SFTS patients. Elevated IL-15 and eotaxin in SFTS patients were reported firstly. The highest levels of pro-inflammatory and anti-inflammatory cytokines coexisted in fatal patients during the first week. Inflammatory mediators remained high levels when death occurred in fatal patients, they were recovered within three weeks in nonfatal patients. Our results showed the occurrence of inflammatory storm in SFTS patients were associated with the severity of SFTS. RANTES and PDGF were down regulated and remained significantly lower levels in fatal patients throughout the course of disease, the concentrations of RANTES and PDGF were remarkably positively correlated with the platelet count. Our results demonstrated that dysregulated inflammatory response was associated with disease pathogenesis and mortality in SFTS patients.

Mutations in the GyrA subunit of DNA gyrase and the ParC subunit of topoisomerase IV in clinical strains of fluoroquinolone-resistant Shigella in Anhui, China.

In this research 26 Shigella isolates were examined by PCR and direct nucleotide sequencing for genetic alterations in the quinolone-resistance determining regions (QRDRs). We tested for the presence of qnr genes by PCR in 91 strains, but no qnr genes were found. The results did show, however, some novel mutations at codon 83 of gyrA (Ser-->Ile) and codon 64 of parC (Ala64-->Cys, Ala64-->Asp), which were related to fluroquinolone resistance.

Galleria mellonella as an in vivo model for assessing the efficacy of antimicrobial agents against Enterobacter cloacae infection.

BACKGROUND/PURPOSE: Enterobacter cloacae is a well-recognized nosocomial pathogen. Use of a rapid, in vivo infection model for E. cloacae that can determine the efficacy of antibiotic therapies could help facilitate screening for new treatments. Nonmammalian model systems of infection, such as Galleria mellonella, have significant logistical and ethical advantages over mammalian models. MATERIALS AND METHODS: We utilized G. mellonella larvae to determine the utility of this infection model to study antibacterial efficacy. G. mellonella killing with heat-killed or live clinical isolates (E. cloacae GN1059 and GN0791) was tested. We also investigated the effect of postinoculation incubation temperature on the survival of infected larvae. The protection of administration of antibiotics to infected larvae was investigated. Finally, we determined the G. mellonella hemolymph burden of E. cloacae after administration of different antibiotics. RESULTS: With live bacterial inocula, G. mellonella killing was significantly dependent on the number of E. cloacae cells injected in a dose-dependent manner. Further, we observed that survival was reduced with increasing the postinoculation temperature. Treatment of a lethal E. cloacae infection with antibiotics that had in vitro activity significantly prolonged the survival of larvae compared with treatment with antibiotics to which the bacteria were resistant. The therapeutic benefit arising from administration of antibiotic correlated with a reduced burden of E. cloacae cells in the hemolymph. CONCLUSION: The G. mellonella infection model has the potential to be used to facilitate the in vivo study of host-pathogen interactions in E. cloacae and the efficacy of antibacterial agents.

Increase in the Prevalence of Resistance Determinants to Trimethoprim/Sulfamethoxazole in Clinical Stenotrophomonas maltophilia Isolates in China.

AIMS: This study was carried to reveal the genetic mechanisms of trimethoprim/sulfamethoxazole (SXT) resistance. METHODS: Among 300 clinical Stenotrophomonas maltophilia isolates from China, resistance determinants such as sul and dfrA genes, integrons and transposase were examined using PCR, DNA sequencing and thermal asymmetric interlaced PCR (TAIL-PCR). Data were analyzed using SPSS 20.0. RESULTS: Of the 300 isolates, 116 (38.7%) were resistant to SXT. An alarming trend of increased resistance to SXT were found over the 10-year period. The positive rates of sul and class 1 integrase (intI1) increased gradually with the development of SXT resistance over the 10-year period. Multiple logistic regression analyses indicated that the genes of qacEΔ1-sul1 (81% vs 46.2%, p = 0.000), sul2 (50.9% vs 9.8%, p = 0.000), intI1 (83.6% vs 65.8%, p = 0.000), dfrA12 (25% vs 3.3%, p = 0.000), dfrA17 (15.5% vs 3.8%, p = 0.000) and dfrA27 (4.3% vs 1.6%, p = 0.01) were more prevalent in SXT-resistant isolates than SXT-susceptible isolates except dfrA1(p = 0.83) and dfrA5(p = 0.18). Sequencing data revealed 12 types of resistance gene cassettes (aar-3-dfrA27, dfrA12-aadA2, dfrA17-aadA5, cmlA1, aacA4, aadA5, arr-3-aacA4, aadA1, aadB-aadA4, aacA4-catB8-aadA1, aadB-aac(6')-II-blaCARB-8 and aac(6')-II-blaCARB-8) located in the class 1 integron in 163 isolates (87% SXT-resistant vs 33.7% SXT-susceptible isolates, p = 0.000). A novel finding was the aar-3-dfrA27 (KC748137) gene cassette. The gene of sul2 linked to transposase in 50 SXT- resistant and 7 SXT- susceptible isolates was detected by TAIL-PCR. CONCLUSIONS: The findings demonstrated a higher prevalence of sul, dfrA, intI1 and resistance gene cassettes in class 1 integron in SXT-resistant clinical S. maltophilia isolates in China. The sul1 and dfrA genes located in integrons and the sul2 linked to transposase may imply wide and rapid dissemination of resistance gene in bacteria.

Susceptibility of Stenotrophomonas maltophilia clinical strains in China to antimicrobial combinations.

We aimed to investigate the activity levels of several combinations of antimicrobials against Stenotrophomonas maltophilia. In this study, the antimicrobial susceptibility of S. maltophilia clinical isolates was determined, and the synergistic activity of three pairs of antimicrobial combinations was evaluated by the fractional inhibitory concentration index (FICI). The antimicrobial susceptibility in vitro against 83 S. maltophilia strains was greater for minocycline (80·7%) than for trimethoprim-sulfamethoxazole (51·8%), and levofloxacin (50·6%). The rate of resistance was highest for ticarcillin-clavulanate and ceftazidime (63·8%) and resistance to trimethoprim-sulfamethoxazole (TMP-SMX) was 48·2%. All three combinations were tested against susceptible isolates. Two of the combinations, TMP-SMX+ceftazidime and levofloxacin+ceftazidime were more effective than the combination of TMP-SMX+levofloxacin. We recommend acquiring more clinical data in order to explore combination therapy, which is a promising treatment of S. maltophilia infections.

Detection of plasmid-mediated quinolone resistance determinants and the emergence of multidrug resistance in clinical isolates of Shigella in SiXian area, China.

A total of 123 Shigella isolates were collected from SiXian area in Anhui, China. Screening was carried out by polymerase chain reaction (PCR) amplification of plasmid-mediated quinolone resistance (PMQR) determinants. Different ß-blactamases genes, plasmid-borne bla(AmpC), 16S rRNA methylase genes, integrons, and mutations in quinolone resistance-determining regions were analysed by PCR for the PMQR-positive isolates.

Comparative study of the mutant prevention concentrations of vancomycin alone and in combination with levofloxacin, rifampicin and fosfomycin against methicillin-resistant Staphylococcus epidermidis.

No mutant-prevention concentration (MPC) with methicillin-resistant Staphylococcus epidermidis (MRSE) has been reported. The study aimed to evaluate the propensity of vancomycin individually and in combination to prevent MRSE from mutation. A total of 10 MRSE clinical isolates were included in the study. Susceptibility testing demonstrated that the susceptibility rates to vancomycin, rifampicin, levofloxacin and fosfomycin were 100, 100, 50 and 90%, respectively. The fractional inhibition concentration indices (FICI) for vancomycin combined with rifampicin, levofloxacin or fosfomycin were ≥1.5 but ≤2, ≥1.5 but ≤2, and >0.5 but ≤1.5, respectively, implying indifferent interactivity. The MPC with susceptible strains was determined to be the lowest antibiotic concentration inhibiting visible growth among 10(10) CFU on four agar plates (9 cm in diameter) after a 72-h incubation at 37°C. The MPCs were 16~32, >64, ≥64 and 4~16 µg ml(-1) for vancomycin, rifampicin, fosfomycin and levofloxacin, respectively. The vancomycin MPCs of combinations with fosfomycin (32 µg ml(-1)), levofloxacin (2 µg ml(-1)) and rifampicin (2 or 4 µg ml(-1)) were 1~4, 16~32 and 16~32 µg ml(-1), respectively. Against mutants selected by vancomycin, rifampicin, levofloxacin and fosfomycin individually, antibiotics had standard MICs of 2~4 µg ml(-1) for vancomycin, >64 µg ml(-1) for rifampicin, 4~8 µg ml(-1) for levofloxacin and 64 µg ml(-1) for fosfomycin. Thus single-step mutation can lead to resistance of MRSE to rifampicin, levofloxacin and fosfomycin, rather than non-susceptibility to vancomycin. Vancomycin-fosfomycin combination might be a superior alternative to vancomycin in blocking the growth of MRSE mutants, especially for high-organism-burden infections.

Vancomycin MICs of the resistant mutants of S. aureus ATCC43300 vary based on the susceptibility test methods used.

Clinical failures with vancomycin against meticillin-resistant Staphylococcus aureus (MRSA) infections have challenged vancomycin's standing as a first-line antimicrobial for these infections. Conventional MIC tests were not predictive of the in vivo therapeutic effect of vancomycin. Thus, we tested the susceptibility for the resistant mutants in the mutant selection window of S. aureus ATCC43300 (a MRSA strain) by three different MIC-testing methods in this paper. The MIC of vancomycin was estimated at 2 µg ml⁻¹ on the Mueller-Hinton agar (MHA) plate only for the resistant mutant that was selected from the plate of vancomycin concentration 12 µg ml⁻¹. The obvious changes of susceptibility testing were found between the resistant mutants and S. aureus ATCC43300 on the Brain-Heart Infusion Agar (BHIA) plates. There were subtle changes in the MIC trend within the susceptible range with the result of Etest for the resistant mutants. The susceptibility for the subcultures of resistant mutants would fall back when the external drug environment disappeared. In comparison with the S. aureus ATCC43300, sequence analysis revealed that there were no mutations in the staphylococcal protein A (spa) sequencing of the resistant mutants. The spa tape is t421 for all isolates.

Stenotrophomonas maltophilia resistance to trimethoprim/sulfamethoxazole mediated by acquisition of sul and dfrA genes in a plasmid-mediated class 1 integron.

Stenotrophomonas maltophilia is becoming a more and more common cause of infections. In this study, the minimal inhibitory concentrations of trimethoprim/sulfamethoxazole (SXT), ceftazidime, minocycline, levofloxacin, chloramphenicol and ticarcillin/clavulanic acid were determined and the distribution of integrons and sul1, sul2 and dfrA genes was investigated in 102 S. maltophilia isolates collected from patients treated in 31 hospitals in Anhui, China, in the month of September in 2006-2008. The rate of resistance to SXT was up to 30.4%, and 64.7% of isolates were class 1 integron-positive. Sequencing data revealed the following novel gene cassettes embedded in class 1 integrons: dfrA17-aadA5; dfrA12-aadA2; aacA4-catB8-aadA1; aadB-aac(6')-II-bla(CARB-8); and arr-3-aacA4. This is the first report of the gene cassettes dfrA17-aadA5 and dfrA12-aadA2 and of sul2 genes in SXT-resistant S. maltophilia isolates in China. None of the SXT-susceptible S. maltophilia isolates were positive for sul2 or dfrA gene products by polymerase chain reaction (PCR), but PCR products for sul1 were detected in 27 SXT-susceptible and 25 SXT-resistant isolates. The findings from this study indicate that the sul1 gene, in combination with dfrA17 and dfrA12 gene cassettes and sul2 genes located within a 7.3kb plasmid, lead to a high rate of SXT resistance and also confirm the need for ongoing resistance surveillance.

ACT-6, a novel plasmid-encoded class C ß-lactamase in a Klebsiella pneumoniae isolate from China.

The purpose of this study was to investigate the phenotypic and molecular characterization of a novel plasmid-mediated AmpC-type ß-lactamase in Klebsiella pneumoniae E701 isolated from Anhui province in China. In comparison with the ACT-1, sequence analysis revealed that there were 43 point mutations in the coding gene, and 10 of which led to amino-acid substitution. Resistance could be transferred by conjugation or transformation with plasmid DNA into E. coli JM109, which was due to the production of a ß-lactamase with an isoelectric point of 8.4 named ACT-6. Cloning, expression, purification and kinetics were carried out to study the characterization of the novel AmpC-type ß-lactamase. The results of MIC determinations and substrate profiles showed there was no significant difference in the activities of the novel enzyme and ACT-1. Moreover, the class 1 integron and the whole open reading frame of the novel AmpC-type ß-lactamase from K.pneumoniae E701 were detectable in the same size plasmid. This is the first report on the emergence of the novel ACT-6 type ß-lactamases in K. pneumoniae.