High Rates of Human Fecal Carriage of mcr-1-Positive Multidrug-Resistant Enterobacteriaceae Emerge in China in Association With Successful Plasmid Families.

Background: mcr-1-mediated colistin resistance in Enterobacteriaceae is concerning, as colistin is used in treating multidrug-resistant Enterobacteriaceae infections. We identified trends in human fecal mcr-1-positivity rates and colonization with mcr-1-positive, third-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae in Guangzhou, China, and investigated the genetic contexts of mcr-1 in mcr-1-positive 3GC-R strains. Methods: Fecal samples were collected from in-/out-patients submitting specimens to 3 hospitals (2011-2016). mcr-1 carriage trends were assessed using iterative sequential regression. A subset of mcr-1-positive isolates was sequenced (whole-genome sequencing [WGS], Illumina), and genetic contexts (flanking regions, plasmids) of mcr-1 were characterized. Results: Of 8022 fecal samples collected, 497 (6.2%) were mcr-1 positive, and 182 (2.3%) harbored mcr-1-positive 3GC-R Enterobacteriaceae. We observed marked increases in mcr-1 (0% [April 2011] to 31% [March 2016]) and more recent (since January 2014; 0% [April 2011] to 15% [March 2016]) increases in human colonization with mcr-1-positive 3GC-R Enterobacteriaceae (P < .001). mcr-1-positive 3GC-R isolates were commonly multidrug resistant. WGS of mcr-1-positive 3GC-R isolates (70 Escherichia coli, 3 Klebsiella pneumoniae) demonstrated bacterial strain diversity; mcr-1 in association with common plasmid backbones (IncI, IncHI2/HI2A, IncX4) and sometimes in multiple plasmids; frequent mcr-1 chromosomal integration; and high mobility of the mcr-1-associated insertion sequence ISApl1. Sequence data were consistent with plasmid spread among animal/human reservoirs. Conclusions: The high prevalence of mcr-1 in multidrug-resistant E. coli colonizing humans is a clinical threat; diverse genetic mechanisms (strains/plasmids/insertion sequences) have contributed to the dissemination of mcr-1, and will facilitate its persistence.

Characterization of CTX-M-140, a Variant of CTX-M-14 Extended-Spectrum ß-Lactamase with Decreased Cephalosporin Hydrolytic Activity, from Cephalosporin-Resistant Proteus mirabilis.

CTX-M-140, a novel CTX-M-type extended-spectrum ß-lactamase (ESBL), was identified in cephalosporin-resistant clinical isolates of Proteus mirabilis CTX-M-140 contained an alanine-to-threonine substitution at position 109 compared to its putative progenitor, CTX-M-14. When it was expressed in an Escherichia coli isogenic background, CTX-M-140 conferred 4- to 32-fold lower MICs of cephalosporins than those with CTX-M-14, indicating that the phenotype was attributable to this single substitution. For four mutants of CTX-M-14 that were constructed by site-directed mutagenesis (A109E, A109D, A109K, and A109R mutants), MICs of cephalosporins were similar to those for the E. coli host strain, which suggested that the alanine at position 109 was essential for cephalosporin hydrolysis. The kinetic properties of native CTX-M-14 and CTX-M-140 were consistent with the MICs for the E. coli clones. Compared with that of CTX-M-14, a lower hydrolytic activity against cephalosporins was observed for CTX-M-140. blaCTX-M-140 is located on the chromosome as determined by I-CeuI pulsed-field gel electrophoresis (I-CeuI-PFGE) and Southern hybridization. The genetic environment surrounding blaCTX-M-140 is identical to the sequence found in different plasmids with blaCTX-M-9-group genes among the Enterobacteriaceae Genome sequencing and analysis showed that P. mirabilis strains with blaCTX-M-140 have a genome size of ∼4 Mbp, with a GC content of 38.7% and 23 putative antibiotic resistance genes. Our results indicate that alanine at position 109 is critical for the hydrolytic activity of CTX-M-14 against oxyimino-cephalosporins.

NDM-1-Producing Citrobacter freundii, Escherichia coli, and Acinetobacter baumannii Identified from a Single Patient in China.

We identified New Delhi metallo-ß-lactamase (NDM-1)-producing Citrobacter freundii GB032, Escherichia coli GB102, and Acinetobacter baumannii GB661 in urine and stool samples from a single patient in China. Plasmid profiling and Southern blotting indicated that blaNDM-1 from GB032 and that from GB102 were likely located on the same plasmid, while blaNDM-1 from GB661 was located on a very large (>400-kb) plasmid. This case underscores the broad host range of blaNDM-1 and its potential to spread between members of the family Enterobacteriaceae and A. baumannii.

Attenuated TLRs in middle ear mucosa contributes to susceptibility of chronic suppurative otitis media.

The variability in the recovery of otitis media (OM) is not well understood. Recent data have shown a critical role for toll-like receptors (TLRs) in inflammatory responses to bacteria. It remains unclear whether TLRs-mediated mucosal immunity plays a role in the OM recovery. The etiology, pathological profile, expression levels of TLR2, TLR4, TLR5, TLR9 and proinflammatory cytokines were measured in human middle-ear mucosae sampled from three subject groups: non-OM group, chronic otitis-media (COM) group, and chronic suppurative otitis-media (CSOM) group. Of the 72 ears, 86.11% CSOM patients were positive for bacteria. The cellular makeup of the middle ear mucosa differs among the three groups. Mucosae from the CSOM group presented chronic inflammation or suppurative inflammation in the rudimentary stroma, mainly with infiltration of monocytes and macrophages. The mRNA and protein levels of TLR2, TLR4, and TLR5 exhibited no difference between the non-OM and COM groups but were significantly lower in the CSOM group. Conversely, there was no significant difference in the TLR9 level among the three groups. Furthermore, proinflammatory cytokines TNF-α, IL-1ß, IFN-γ, IL-6 were up-regulated in the CSOM group. This study provides evidence that the variability in clinical otitis media recovery might be associated with the variability in the expression of mucosal TLRs. Reduced TLR levels in the middle-ear mucosa might cause weak host response to bacteria, persistent inflammation and susceptibility to CSOM.

[Differential expression of toll-like receptors in chronic suppurative otitis media and cholesteatoma].

OBJECTIVE: To investigate the differential expression of toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4) and their potential role in the pathogenesis of chronic suppurative otitis media and cholesteatoma. METHODS: Normal canal skin of 30 patients with tympanosclerosis were enrolled as control, 30 cases with chronic suppurative otitis media and 30 patients with cholesteatoma were studied. Real-time PCR, Western blot and Immunohistochemistry were preformed to detect the expression of TLR2/TLR4 in normal canal skin, mucosa and granulation tissue of chronic suppurative otitis media, mucosa, granulation tissue, cholesteatoma epithelium of cholesteatoma, and the differential expression were analyzed. RESULTS: (1) the mRNA and protein expression of TLR2 and TLR4 were detected in all normal canal skin, mucosa and granulation tissue of chronic suppurative otitis media, mucosa, granulation tissue, cholesteatoma epithelium of cholesteatoma. (2) Both mRNA and protein level of TLR2/TLR4 in mucosa of chronic suppurative otitis media and cholesteatoma were higher than those in normal canal skin, but lower in cholesteatoma epithelium, there was no significant difference in mucosa of the two otitis media groups. (3) The mRNA and protein expression of TLR2/TLR4 in granulation tissue of chronic suppurative otitis media and cholesteatoma were significant increased when compared with normal canal skin, and TLR2 expression level was higher in granulation tissue of cholesteatoma than in chronic suppurative otitis media. (4) TLR2/TLR4 positive cells mainly infiltrated in granulations, significantly more than in normal skin, while fewer in the epithelium of cholesteatoma. CONCLUSIONS: Differential expression of TLR2 and TLR4 in mucosa suggests middle ear is a TLR2/TLR4 participated functional modulation of the innate immune system and also suggests that they may play a different role in the pathophysiology of chronic otitis media and cholesteatoma.

[Effect of garlicin on the serum levels of interleukin 4 and interferon gamma in allergic rhinitis model in rats].

OBJECTIVE: To investigate the effect of garlicin on the levels of interferon gamma (INF-gamma) and interleukin 4 (IL-4) in blood of allergic rhinitis rat model. METHODS: Thirty healthy female SD rats were randomly divided into 3 groups: control group, negative control group and experimental group, 10 rats for each group. Ten rats (experimental group) were sensitized and intranasally challenged by ovalbumin, aluminium hydroxide hydrate gel and Bordetella pertussis inactive microorganism suspension adjuvants, as allergic rhinitis models, and then injection of garlicin(0.4 ml) intraperitoneally per day for 10 days. Control group rats were immunized as experimental group, and then injection of physiological saline as equal volume as garlicin. Negative control group rats were investigated using physiological saline. Blood of intrajugular vein of rat was extracted for separated plasma Enzyme liked immunosorbent assay (ELISA) was utilized to detect the serum levels of IL-4 and IFN-gamma. RESULTS: The serum levels (x +/- s) of IL4 were (22.81 +/- 8.79) pg/L, (41.43 +/- 4.93) pg/L, (9.93 +/- 2.07) pg/L, and those of IFN-gamma were (22.32 +/- 11.20) pg/L, (11.35 +/- 2.45) pg/L and (21.69 +/- 5.93) pg/L, respectively, among experimental group, control group and negative control group. The serum level of IL-4 in experimental group rats was lower than value of control group rats (t = 3.22, P < 0.05), while higher than negative control group (t = 4.17, P < 0.05). The serum level of IFN-gamma was increased significantly in experimental group rats with significant difference when compared with value of control group rats (t = 3.84, P < 0.05), while no difference was shown between experimental group and negative control group (t = 1.47, P > 0.05). CONCLUSIONS: Garlicin could increase serum level of INF-gamma and decrease serum level of IL4 significantly in allergic rhinitis rat model. It played an important role on regulating serum levels of cytokines of Thl and Th2.

[Relationship between transcription factor GATA-3 and cytokines expression in chronic sinusitis].

OBJECTIVE: To explore the correlation between the expression of GATA-3 and the level of local cytokines (IL-5, IL-6 and IL-8). METHODS: The levels of IL-5, IL-6 and IL-8 in ethmoid sinus mucosa were titrated in 45 patients with chronic rhinosinusitis and 11 normal subjects by ELISA. Patients were divided into AR group (with allergic rhinitis) and NAR group (without allergic rhinitis) . Semi-quantitative RT-PCR and immunohistochemical staining were used to examine the GATA-3 expression in nasal mucosa. The correlation between the expression of GATA-3 and the levels of cytokines was evaluated. RESULTS: IL-5, IL-6 and IL-8 levels in both AR and NAR groups were significantly elevated compared with normal group (all P < 0.01 for AR group; P < 0.05, 0.05, 0.01 for NAR group, respectively), and they were much higher in AR group in comparison with NAR group (P < 0.01, 0.05, 0.01, respectively). Semi-quantitative RT-PCR showed that AR and NAR groups had markedly greater level of GATA-3 mRNA than that in control group (P < 0.01, respectively), and the level of GATA-3 mRNA in AR group was further higher than that in NAR group (P < 0.01). Immunohistochemical staining illustrated that GATA-3 was primarily presented in cytoplasma and the GATA-3 positive cells were mainly infiltrating inflammatory cells in submucosa. The mean GATA-3 positive-staining rate was (27. 90 +/- 16.75)% and (10.22 +/- 0.05)% in AR and NAR group, which were markedly higher than (1.30 +/- 1.78)% in control group (P < 0.01, respectively). Pearson correlation analysis demonstrated that GATA-3 positive-staining rate was closely correlated with IL-5 level, but not IL-6 and IL-8. The correlation coefficient was 0. 712 for GATA-3 and IL-5 (P < 0.01), 0.200 for GATA-3 and IL-6 (P > 0.05), 0.089 for GATA-3 and IL-8 (P > 0.05). CONCLUSIONS: Activation of GATA-3 might be one of the mechanisms for induction of IL-5 expression in chronic rhinosinusitis . Concomitance of allergic rhinitis with chronic rhinosinusitis further increased expression of GATA-3, and subsequently enhanced IL-5 expression. Chronic sinusitis may be related to allergy, and GATA-3 may play a key role in the pathogenesis of chronic sinusitis.