Objective To investigate the phenotypic and genetic characteristics of the lysostaphin‐resistant Staphylococcus aureus variants induced by recombinant lysostaphin in vitro .Methods Three clinical isolates of S . aureus ,including two resistant to methicillin (MRSA ) and one susceptible to methicillin (MSSA ) were induced by treatment with sub‐MIC of recombinant lysostaphin via one‐step selection in vitro .Susceptibility of the variants to antibiotics were determined and compared with their parental strains .The full length of femABX genes was amplified by polymerase chain reaction and sequenced to identify the potential mutation sites in these genes .The growth‐curve in liquid medium and virulence in a mouse systemic infection model of both parental and variant strains were observed . Results The frequency of lysostaphin resistance in S . aureus was between 10-4 to 10-8 following induction by lysostaphin . Resistance to lysostaphin was associated with a significant decrease in growth rate in vitro and virulence in vivo ,as well as increased susceptibility toβ‐lactams evidenced by the M IC of β‐lactams against the variants as low as 1/4 000 to 1/2 of the M IC against their parental strains . Sequencing of f emA BX genes showed mutation in femA gene in both variants ,which resulted in a premature termination codon .Conclusions Resistance of S . aureus to lysostaphin may develop following induction by recombinant lysostaphin in vitro . The lysostaphin‐resistant S . aureus variants are characteristic of lower growth rate , decreased virulence ,and higher susceptibility to β‐lactams .

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 µg/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(6')-Ib, aac(6')-II, aph(4)-Ia, aph(3')-I, aph(3')-IIb, aph(3')-IIIa, aph(3')-VIa, aph(2″)-Ib, aph(2″)-Ic, aph(2″)-Id, ant(2″)-Ia, ant(3″)-I and ant(4')-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(6')-Ib, detection rate of 45.10%; aph(3')-I, detection rate of 47.06%; aph(3')-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(6')-Ib+ant(3″)-I+armA (detection rate of 25.49%), aac(3)-I+aph(3')-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(3')-I and aac(6')-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.

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 and activity of d-serine alone and in combination with -lactams against methicillin-resistant (MRSA) strains, and to explore the possible sensitization mechanisms. The activity of d-serine, -lactams alone and in combinations was evaluated both by standard MICs, time-kill curves and checkerboard assays, and by murine systemic infection model as well as neutropenic thigh infection model. An synergistic effect was demonstrated with the combination of d-serine and -lactams against MRSA standard and clinical strains. Importantly, the combinations enhanced the therapeutic efficacy in the animal models as compared to -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 -lactams against MRSA strains both and . Considering the relatively good safety of d-serine alone or in combination with -lactams, d-serine is worth following up as new anti-MRSA infection strategies.

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 S₂ for 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 IS26 mediated intramolecular transposition was found in Mut-S, which led to mgrB truncation, lipid A modification and hence S₂ resistance. 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.