Polymyxin resistance caused by large-scale genomic inversion due to IS26 intramolecular translocation in Klebsiella pneumoniae

Polymyxin B and polymyxin E(colistin)are presently considered the last line of defense against human infections caused by multidrug-resistant Gram-negative organisms such as carbapenemase-producer Enterobacterales,Acinetobacter baumannii,and Klebsiella pneumoniae.Yet resistance to this last-line drugs is a major public health threat and is rapidly increasing.Polymyxin S2(S2)is a polymyxin B analogue previously synthesized in our institute with obviously high antibacterial activity and lower toxicity than polymyxin B and colistin.To predict the possible resistant mechanism of S2for wide clinical application,we experimentally induced bacterial resistant mutants and studied the preliminary resistance mechanisms.Mut-S,a resistant mutant of K.pneumoniae ATCC BAA-2146(Kpn2146)induced by S2,was analyzed by whole genome sequencing,transcriptomics,mass spectrometry and complementation experiment.Surprisingly,large-scale genomic inversion(LSGI)of approximately 1.1 Mbp in the chromosome caused by IS26mediated intramolecular transposition was found in Mut-S,which led to mgrB truncation,lipid A modification and hence S2resistance.The resistance can be complemented by plasmid carrying intact mgrB.The same mechanism was also found in polymyxin B and colistin induced drug-resistant mutants of Kpn2146(Mut-B and Mut-E,respectively).This is the first report of polymyxin resistance caused by IS26 intramolecular transposition mediated mgrB truncation in chromosome in K.pneumoniae.The findings broaden our scope of knowledge for polymyxin resistance and enriched our understanding of how bacteria can manage to survive in the presence of antibiotics.

In vitro and in vivo activity of D-serine in combination with β-lactam antibiotics against methicillin-resistant Staphylococcus aureus

As D-amino acids play important roles in the physiological metabolism of bacteria,combination of D-amino acids with antibiotics may provide synergistic antibacterial activity. The aim of the study was to evaluate in vitro and in vivo activity of D-serine alone and in combination withβ-lactams against methicillin-resistant Staphylococcus aureus(MRSA) strains, and to explore the possible sensitization mechanisms. The activity of D-serine, β-lactams alone and in combinations was evaluated both in vitro by standard MICs, time–kill curves and checkerboard assays, and in vivo by murine systemic infection model as well as neutropenic thigh infection model. An in vitro synergistic effect was demonstrated with the combination of D-serine and β-lactams against MRSA standard and clinical strains.Importantly, the combinations enhanced the therapeutic efficacy in the animal models as compared toβ-lactam alone groups. Initial mechanism study suggested possible revision of D-alanine-D-alanine residue to D-alanine-D-serine in peptidoglycan by adding of D-alanine in the medium, which may cause decreased affinity to PBPs during transpeptidation. In conclusion, D-serine had synergistic activity in combination with β-lactams against MRSA strains both in vitro and in vivo. Considering the relatively good safety of D-serine alone or in combination with β-lactams, D-serine is worth following up as new anti-MRSA infection strategies.

Genetic basis of high level aminoglycoside resistance in Acinetobacter baumannii from Beijing,China

The objective of this study was to investigate the genetic basis of high level aminoglycoside resistance in Acinetobacter baumannii clinical isolates from Beijing,China.173 A.baumannii clinical isolates from hospitals in Beijing from 2006 to 2009 were first subjected to high level aminoglycoside resistance(HLAR,MIC to gentamicin and amikacin>512 mg/mL)phenotype selection by broth microdilution method.The strains were then subjected to genetic basis analysis by PCR detection of the aminoglycoside modifying enzyme genes(aac(3)-Ⅰ,aac(3)-Ⅱc,aac(60)-Ⅰb,aac(60)-Ⅱ,aph(4)-Ⅰa,aph(30)-Ⅰ,aph(30)-Ⅱb,aph(30)-Ⅲa,aph(30)-Ⅵa,aph(2″)-Ⅰb,aph(2″)-Ⅰc,aph(2″)-Ⅰd,ant(2″)-Ⅰa,ant(3″)-Ⅰand ant(40)-Ⅰa)and the 16S rRNA methylase genes(armA,rmtB and rmtC).Correlation analysis between the presence of aminoglycoside resistance gene and HLAR phenotype were performed by SPSS.Totally 102(58.96%)HLAR isolates were selected.The HLAR rates for year 2006,2007,2008 and 2009 were 52.63%,65.22%,51.11%and 70.83%,respectively.Five modifying enzyme genes(aac(3)-Ⅰ,detection rate of 65.69%;aac(60)-Ⅰb,detection rate of 45.10%;aph(30)-Ⅰ,detection rate of 47.06%;aph(30)-Ⅱb,detection rate of 0.98%;ant(3″)-Ⅰ,detection rate of 95.10%)and one methylase gene(armA,detection rate of 98.04%)were detected in the 102 A.baumannii with aac(3)-Ⅰ+aac(60)-Ⅰ+þant(3″)-Ⅰ+armA(detection rate of 25.49%),aac(3)-Ⅰ+aph(30)-Ⅰ+ant(3″)-Ⅰ+armA(detection rate of 21.57%)and ant(3″)-Ⅰ+armA(detection rate of 12.75%)being the most prevalent gene profiles.The values of chi-square tests showed correlation of armA,ant(3″)-Ⅰ,aac(3)-Ⅰ,aph(30)-Ⅰand aac(60)-Ⅰb with HLAR.armA had significant correlation(contingency coefficient 0.685)and good contingency with HLAR(kappa 0.940).The high rates of HLAR may cause a serious problem for combination therapy of aminoglycoside with β-lactams against A.baumannii infections.As armA was reported to be able to cause high level aminoglycoside resistance to most of the clinical important aminoglycosides(gentamicin,amikacin,tobramycin,etc),the function of aminoglycoside modifying enzyme gene(s)in A.baumannii carrying armA deserves further investigation.