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.

Sub-inhibitory concentrations of fluoroquinolones increase conjugation frequency.

Bacteria are subjected to sub-minimal inhibitory concentrations (sub-MIC) of antibiotics in various niches where the low-dosage treatment plays a key role in antibiotic resistance selection. However, the mechanism of sub-MIC of antibiotics on the resistant gene transfer is largely unknown. Here, we used Escherichia coli SM10λpir in which the RP4 plasmid was chromosomally-integrated as the donor strain, to investigate the effects of sub-MIC of Ciprofloxacin(Cip) or Levofloxacin(Lev) on conjugational transfer of mobilisable plasmid-pUCP24T from SM10λpir to Pseudomonas aeruginosa. The results showed that the transfer frequency was significantly increased by treating E. coli with sub-MIC of Cip or Lev. To investigate the molecular mechanisms, complete transcriptome sequencing was performed. We found that the sub-MIC of Cip or Lev enhanced the expression of several genes on the RP4 plasmid, which was consistent with the conjugation efficiency. Moreover, the expression of genes associated with SOS response in donor SM10λpir was increased, but had no correlation with conjugation efficiency. These findings suggested that sub-MIC of Cip or Lev may promote conjugational transfer by up-regulating the expression of conjugation associated genes via an SOS-independent mechanism.

Serum IFN-γ-inducible chemokines CXCL9 and CXCL10 are elevated in non-immediate drug hypersensitivity reactions.

BACKGROUND: The recruitment to the skin of drug-responsive T cells is responsible for the inflammatory profiles of non-immediate drug hypersensitivity reactions (niDHRs). Maculopapular exanthema (MPE) and Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN) have quite distinct T cell infiltrating patterns. OBJECTIVE: To investigate serum levels of CXCL9, CXCL10 and IFN-γ in patients with niDHRs, including MPE and SJS/TEN, to evaluate correlations between the cytokines, and to determine whether the inflammatory factors correlate with clinical severity in patients with SJS/TEN. METHOD: Twenty-four patients with SJS/TEN, 24 patients with MPE, and 24 healthy donors with good tolerance to the drugs involved in the drug reactions were recruited into the study. The modified severity-of-illness score for TEN (SCORTEN) and detachment of body surface area (dBSA) were used to assess the clinical severity of SJS/TEN. Serum levels of CXCL9, CXCL10 and IFN-? were determined by ELISA. RESULTS: The niDHRs group, SJS/TEN and MPE subgroups all exhibited significantly higher levels of CXCL9, CXCL10 and IFN-γ compared with the control group (P < 0.001). Serum IFN-γ levels were positively correlated with CXCL9 levels and CXCL10 levels in patients with niDHRs (rs = 0.576, rs = 0.449, P < 0.05). None of the levels of CXCL9, CXCL10 and IFN-γ had any correlation with modified SCOTEN index or dBSA in SJS/TEN group. CONCLUSIONS: The results suggest Th1 cytokine IFN-γ and chemokines CXCL9 and CXCL10 may play roles in the pathogenesis of niDHRs.

Impact of an Intervention to Control Imipenem-Resistant Acinetobacter baumannii and Its Resistance Mechanisms: An 8-Year Survey.

BACKGROUND: This study aimed to examine the impact of an intervention carried out in 2011 to combat multi-drug resistance and outbreaks of imipenem-resistant Acinetobacter baumannii (IRAB), and to explore its resistance mechanism. METHODS: A total of 2572 isolates of A. baumannii, including 1673 IRAB isolates, were collected between 2007 and 2014. An intervention was implemented to control A. baumannii resistance and outbreaks. Antimicrobial susceptibility was tested by calculating minimal inhibitory concentrations (MICs), and outbreaks were typed using pulsed-field gel electrophoresis (PFGE). Resistance mechanisms were explored by polymerase chain reaction (PCR) and whole genome sequencing (WGS). RESULTS: Following the intervention in 2011, the resistance rates of A. baumannii to almost all tested antibiotics decreased, from 85.3 to 72.6% for imipenem, 100 to 80.8% for ceftriaxone, and 45.0 to 6.9% for tigecycline. The intervention resulted in a decrease in the number (seven to five), duration (8-3 months), and departments (five to three) affected by outbreaks; no outbreaks occurred in 2011. After the intervention, only bla AMPC (76.47 to 100%) and bla TEM- 1 (75.74 to 96.92%) increased (P < 0.0001); whereas bla GES- 1 (32.35 to 3.07%), bla PER- 1 (21.32 to 1.54%), bla OXA- 58 (60.29 to 1.54%), carO (37.50 to 7.69%), and adeB (9.56 to 3.08%) decreased (P < 0.0001). Interestingly, the frequency of class B ß-lactamase genes decreased from 91.18% (bla SPM- 1) and 61.03% (bla IMP- 1) to 0%, while that of class D bla OXA- 23 increased to 96.92% (P < 0.0001). WGS showed that the major PFGE types causing outbreaks each year (type 01, 11, 18, 23, 26, and 31) carried the same resistance genes (bla KPC- 1, bla ADC- 25, bla OXA- 66, and adeABC), AdeR-S mutations (G186V and A136V), and a partially blocked porin channel CarO. Meanwhile, plasmids harboring bla OXA- 23 were found after the intervention. CONCLUSION: The intervention was highly effective in reducing multi-drug resistance of A. baumannii and IRAB outbreaks in the long term. The resistance mechanisms of IRAB may involve genes encoding ß-lactamases, efflux pump overexpression, outer membrane porin blockade, and plasmids; in particular, clonal spread of bla OXA- 23 was the major cause of outbreaks. Similar interventions may also help reduce bacterial resistance rates and outbreaks in other hospitals.

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.

Quorum Sensing N-acyl Homoserine Lactones-SdiA Suppresses Escherichia coli-Pseudomonas aeruginosa Conjugation through Inhibiting traI Expression.

Conjugation is a key mechanism for horizontal gene transfer and plays an important role in bacterial evolution, especially with respect to antibiotic resistance. However, little is known about the role of donor and recipient cells in regulation of conjugation. Here, using an Escherichia coli (SM10λπ)-Pseudomonas aeruginosa (PAO1) conjugation model, we demonstrated that deficiency of lasI/rhlI, genes associated with generation of the quorum sensing signals N-acyl homoserine lactones (AHLs) in PAO1, or deletion of the AHLs receptor SdiA in the donor SM10λπ both facilitated conjugation. When using another AHLs-non-producing E. coli strain EC600 as recipient cells, deficiency of sdiA in donor SM10λπ hardly affect the conjugation. More importantly, in the presence of exogenous AHLs, the conjugation efficiency between SM10λπ and EC600 was dramatically decreased, while deficiency of sdiA in SM10λπ attenuated AHLs-inhibited conjugation. These data suggest the conjugation suppression function of AHLs-SdiA chemical signaling. Further bioinformatics analysis, ß-galactosidase reporter system and electrophoretic mobility shift assays characterized the binding site of SdiA on the promoter region of traI gene. Furthermore, deletion of lasI/rhlI or sdiA promoted traI mRNA expression in SM10λπ and PAO1 co-culture system, which was abrogated by AHLs. Collectively, our results provide new insight into an important contribution of quorum sensing system AHLs-SdiA to the networks that regulate conjugation.

Mechanism of azithromycin inhibition of HSL synthesis in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen and a leading cause of nosocomial infections. Unfortunately, P. aeruginosa has low antibiotic susceptibility due to several chromosomally encoded antibiotic resistance genes. Hence, we carried out mechanistic studies to determine how azithromycin affects quorum sensing and virulence in P. aeruginosa. lasI and rhlI single and double mutants were constructed. We then undertook a quantitative approach to determine the optimal concentration of azithromycin and culture time that can affect the expression of HSLs. Furthermore, based on the above results, the effect on quorum sensing was analyzed at a transcriptional level. It was found that 2 µg/mL azithromycin caused a 79% decrease in 3-oxo-C12-HSL secretion during cultivation, while C4-HSL secretion was strongly repressed in the early stages. Azithromycin acts on ribosomes; to determine whether this can elicit alternative modes of gene expression, transcriptional regulation of representative virulence genes was analyzed. We propose a new relationship for lasI and rhlI: lasI acts as a cell density sensor, and rhlI functions as a fine-tuning mechanism for coordination between different quorum sensing systems.