RpoN (sigma factor 54) contributes to bacterial fitness during tracheal colonization of Bordetella bronchiseptica.

The Gram-negative pathogenic bacterium Bordetella bronchiseptica is a respiratory pathogen closely related to Bordetella pertussis, the causative agent of whooping cough. Despite sharing homologous virulence factors, B. bronchiseptica infects a broad range of mammalian hosts, including some experimental animals, whereas B. pertussis is strictly adapted to humans. Therefore, B. bronchiseptica is often used as a representative model to explore the pathogenicity of Bordetella in infection experiments with laboratory animals. Although Bordetella virulence factors, including toxins and adhesins have been studied well, our recent study implied that unknown virulence factors are involved in tracheal colonization and infection. Here, we investigated bacterial genes contributing to tracheal colonization by high-throughput transposon sequencing (Tn-seq). After the screening, we picked up 151 candidate genes of various functions and found that a rpoN-deficient mutant strain was defective in tracheal colonization when co-inoculated with the wild-type strain. rpoN encodes σ54 , a sigma factor that regulates the transcription of various genes, implying its contribution to various bacterial activities. In fact, we found RpoN of B. bronchiseptica is involved in bacterial motility and initial biofilm formation. From these results, we propose that RpoN supports bacterial colonization by regulating various bacteriological functions.

Promoter Variation and Gene Expression of mcr-1-Harboring Plasmids in Clinical Isolates of Escherichia coli and Klebsiella pneumoniae from a Chinese Hospital.

Next-generation sequencing of 6 mcr-1-harboring Escherichia coli and Klebsiella pneumoniae isolates collected from a tertiary care hospital in China revealed significant sequence variations in the regions flanking the mcr-1 gene. While sequence variations significantly affected the expression and promoter activity of mcr-1, the mcr-1 gene expression levels did not correlate with the in vitro colistin resistance levels, which warrants further in-depth investigations.

Direct Detection and Identification of Bacterial Pathogens from Urine with Optimized Specimen Processing and Enhanced Testing Algorithm.

Rapid and accurate detection and identification of microbial pathogens causing urinary tract infections allow prompt and specific treatment. We optimized specimen processing to maximize the limit of detection (LOD) by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and evaluated the capacity of combination of MALDI-TOF MS and urine analysis (UA) for direct detection and identification of bacterial pathogens from urine samples. The optimal volumes of processed urine, formic acid/acetonitrile, and supernatant spotted onto the target plate were 15 ml, 3 µl, and 3 µl, respectively, yielding a LOD of 1.0 × 105 CFU/ml. Among a total of 1,167 urine specimens collected from three hospital centers, 612 (52.4%) and 351 (30.1%) were, respectively, positive by UA and urine culture. Compared with a reference method comprised of urine culture and 16S rRNA gene sequencing, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of MALDI-TOF MS alone and MALDI-TOF MS coupled with UA were 86.6% versus 93.4% (χ2 = 8.93; P < 0.01), 91.5% versus 96.3% (χ2 = 7.06; P < 0.01), 81.5% versus 96.4% (χ2 = 37.32; P < 0.01), and 94.1% versus 93.1% (χ2 = 0.40; P > 0.05), respectively. No significant performance differences were revealed among the three sites, while specificity and NPV of MALDI-TOF MS for males were significantly higher than those for females (specificity, 94.3% versus 77.3%, χ2 = 44.90, P < 0.01; NPV, 95.5% versus 86.1%, χ2 = 18.85, P < 0.01). Our results indicated that the optimization of specimen processing significantly enhanced analytical sensitivity and that the combination of UA and MALDI-TOF MS provided an accurate and rapid detection and identification of bacterial pathogens directly from urine.

SdiA Improves the Acid Tolerance of E. coli by Regulating GadW and GadY Expression.

The acid tolerance mechanism is important for Escherichia coli to resist acidic conditions encountered in mammalian host digestive tract environment. Here, we explored how the LuxR protein SdiA influenced E. coli acid tolerance ability in the context of the glutamate- and glutamine-dependent acid resistance system (AR2). First, using a growth and acid shock assay under different acid stresses, we demonstrated that the deletion of sdiA in SM10λpir or BW25113 led to impaired growth under the acidic environment of pH 3-6, which was restored by complementary expression of SdiA. Next, transcriptome sequencing and qPCR disclosed that the expression of glutamate decarboxylase W (GadW) and GadY, the key members of the AR2 system, were regulated by SdiA. Further, ß-galactosidase reporter assays showed that the promoter activity of gadW and gadY was positively regulated by SdiA. Moreover, qPCR and ß-galactosidase reporter assays confirmed that the regulation of SdiA on GadW, but not GadY, could be enhanced by quorum sensing (QS) signal molecules AHLs. Collectively, these data suggest that SdiA plays a crucial role in acid tolerance regulation of E. coli. Our findings provide new insights into the important contribution of quorum sensing system AHLs-SdiA to the networks that regulate acid tolerance.

Ceftazidime/avibactam Improves the Antibacterial Efficacy of Polymyxin B Against Polymyxin B Heteroresistant KPC-2-Producing Klebsiella pneumoniae and Hinders Emergence of Resistant Subpopulation in vitro.

Due to the increasing multidrug resistance and limited antibiotics, polymyxin B revived as the last resort for the treatment of carbapenemase-producing Klebsiella pneumoniae (CRKP). Unfortunately, the heteroresistance hampers polymyxin B monotherapy treatment via the amplification of resistant subpopulation. Reliable polymyxin B based combinations are demanded. Ceftazidime/avibactam has been regarded as a new salvage therapy against CRKP. The occurrence of heteroresistance was confirmed by population analysis profiling (PAP). Our study demonstrated that polymyxin B and ceftazidime/avibactam combinations improved the in vitro antimicrobial activity of polymyxin B and delayed or suppressed the regrowth of resistant subpopulation by time-kill studies. Ceftazidime/avibactam at around MIC values (0.5-1 × MIC) plus clinically achievable concentrations of polymyxin B (0.5-2 mg/L) resulted in sustained killing against polymyxin B-heteroresistant isolates. Active PmrAB and PhoPQ systems and a pmrA mutation (G53R) in resistant subpopulation might associate with heteroresistance, but further investigation was required. Our findings suggested that the heteroresistance represented barriers to polymyxin B efficacy, and the combination of polymyxin B with ceftazidime/avibactam could be potentially valuable for the treatment of heteroresistant CRKP. Further, in vivo studies need to be performed to evaluate the efficacy of this combination against heteroresistant strains.

High Prevalence of Metallo-ß-Lactamase-Producing Enterobacter cloacae From Three Tertiary Hospitals in China.

Enterobacter cloacae has recently emerged as one of the most common carbapenem-resistant Enterobacteriaceae. The emergence and spread of metallo-ß-lactamase-producing E. cloacae have posed an immediate threat globally. Here, we investigated the molecular characteristics of 84 carbapenem-resistant Enterobacter cloacae (CREL) collected from three tertiary hospitals in China between 2012 and 2016. Species identification and antimicrobial susceptibility testing were performed using a VITEK-2 system. Carbapenems, polymyxins B, and tigecycline were tested by broth microdilution method. The carbapenem in activation method (CIM) and cefoxitin three-dimensional test were used to detect carbapenemase and AmpC ß-lactamase, respectively. Isolates were screened for ß-lactam resistance genes by PCR, and expression of ompC and ompF was determined by qRT-PCR. Genetic relatedness was performed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST), while selected isolates were subjected to whole-genome sequencing. Among the 84 CREL isolates, 50 (59.5%) were detected as carbapenemase producers. NDM-1 was the dominant carbapenemase (80.0%), followed by IMP-26 (8.0%) and IMP-4 (6.0%). Notably, we identified the first NDM-1 and IMP-1 co-producing E. cloacae, carrying plasmids of several incompatibility (Inc) groups, including IncHI2, IncHI2A, and IncN. Most isolates showed decreased expression of ompC and/or ompF, and contained a broad distribution of ESBLs and AmpC ß-lactamases. These findings suggested that different molecular mechanisms, including carbapenemase, ESBL and/or AmpC plus loss of porins, have contributed to carbapenem resistance. The bla NDM-1-harboring plasmids contained highly conserved gene environment around bla NDM-1 (bla NDM-1-ble MBL-trpF-dsbD-cutA1-groES-groEL), which could be associated with the potential dissemination of bla NDM-1. IMP-type MBL was located within a variety of integrons and usually contained various gene cassettes encoding multidrug resistance. These isolates produced 54 different pulsotypes, and were classified into 42 STs by MLST. Nineteen bla NDM-1-positive E. cloacae isolates obtained from Ningxia had the same pulsotype (PFGE type 1), belonging to ST78 within clonal complex 74 (CC74). The plasmid-based replicon typing indicated that IncX3 plasmids mediated the dissemination of bla NDM-1 among these homologous strains. This is the first report on the outbreak of NDM-1-producing E. cloacae ST78 with contribution of IncX3 plasmids in Northwestern China. There's an immediate need to intensify surveillance attentively to prevent and control the further spread of NDM-1 in China.

Regulatory Effect of Catalpol on Th1/Th2 cells in Mice with Bone Loss Induced by Estrogen Deficiency.

PROBLEM: Estradiol (E2 ) deficiency can cause bone loss and the skew of Th1/Th2 cells. However, the correlation between the Th1/Th2 cells and the bone loss induced by estrogen deficiency remains unclear. Our aim was to investigate the role of Th1/Th2 in bone loss induced by estrogen deficiency and elucidated the therapeutical effect of catalpol in this condition. METHOD OF STUDY: Young, sham-operated (Sham), ovariectomized (Ovx), and naturally aged mice, treated with catalpol at different doses or control vehicle, were used in this study as indicated in each experiment. ELISA assay, dual-energy X-ray absorptiometry, and flow cytometry were used to analyze E2 , C-terminal telopeptides of type I collagen (CTx-I), bone mineral density (BMD), and Th1/Th2 subsets, respectively. The mRNA and protein expressions of specific transcription factors for Th1/Th2 cells (T-bet and GATA-3) were analyzed using real-time quantitative PCR and Western blot, respectively. RESULTS: Bone mineral density and E2 levels positively correlated with the proportion of Th2 subset while negatively correlated with that of Th1 subset and the ratio of Th1/Th2. Catalpol alleviated bone loss effectively by regulating Th1/Th2 polarization. Catalpol promoted the expression of Th2-specific transcription factors while inhibited that associated with Th1. CONCLUSION: Th1/Th2 skew is involved in bone loss induced by estrogen deficiency. Catalpol alleviates bone loss effectively by regulating Th1/Th2 paradigm.