Mechanisms of fosfomycin resistance in clinical isolates of carbapenem-resistant Klebsiella pneumoniae.

OBJECTIVES: Older antimicrobials such as fosfomycin are being considered as alternative agents in the treatment of drug-resistant organisms. However, there are limited data on the usefulness of fosfomycin against carbapenem-resistant Klebsiella pneumoniae (CRKP). This study aimed to investigate the prevalence of fosfomycin resistance and associated mechanisms in CRKP. METHODS: A total of 99 clinical CRKP isolates were collected in the Second Affiliated Hospital of Wenzhou Medical University (Wenzhou, China) between January 2017 and June 2018. Fosfomycin susceptibility testing was performed by the agar dilution method. Carbapenemase and fosfomycin resistance genes were detected by PCR. Analysis of the murA, glpT, uhpT, uhpA, ptsI and cyaA genes was performed by PCR and sequencing of four fosfomycin-resistant fosA3-negative CRKP strains. Conjugation experiments were employed to determine the mobility of the fosA3 gene. RESULTS: Of the 99 CRKP isolates, fosfomycin-non-susceptibility was detected in 48 (48.5%) isolates, among which the fosA3 gene was detected in 44. Among the four fosfomycin-resistant fosA3-negative CRKP isolates, one isolate possessed a single nucleotide insertion and deletion mutations as well as 219 nucleotide substitution mutations in murA, two isolates possessed deletion or mutation of large DNA fragments in glpT, and one isolate possessed a fragment insertion sequence in glpT. Transfection into Escherichia coli J53 via plasmid conjugation was successful for 19 (43.2%) of the 44 fosA3-positive CRKP isolates. CONCLUSION: The fosA3 gene is the primary mechanism of fosfomycin resistance in CRKP and can be transmitted widely by plasmid in hospitals. Mutations in murA and glpT were found in fosfomycin-resistant fosA3-negative CRKP.

Regulation of gene expression of hcp, a core gene of the type VI secretion system in Acinetobacter baumannii causing respiratory tract infection.

Purpose. The objective of the current study was to investigate whether hcp plays a role in the process of Acinetobacter baumannii infection and to examine clinically relevant factors that may affect hcp expression.Methodology. Seventy-seven A. baumannii isolates from patients with a respiratory infection at the Second Affiliated Hospital and Yuying Childrens Hospital of Wenzhou Medical University (Wenzhou, China) were included in this study. PCR was performed to screen for the presence of hcp. Quantitative real time polymerase chain reaction (qRT-PCR) was carried out to examine the expression of hcp.Results. A total of 77.9 % (60 of 77) of the A. baumannii clinical isolates possessed the hcp gene. Expression of hcp was found to be strain-specific and associated with the infection status. Higher gene expression of hcp was found for invasive A. baumannii isolates causing an infection relative to the colonization group, and for the same strain at a post-infection status compared with that prior to infection. Acid environment was also found to be a trigger of hcp gene expression.Conclusion. The type VI secretion system and hcp predominate in A. baumannii causing respiratory infections. Expression of hcp is regulated by the infection status and acid environment, and might play a role in the process of triggering infection by the colonizer.

Effect of interspecies quorum sensing on the formation of aerobic granular sludge.

Quorum sensing (QS) is a form of microbial communication that relies on small signal molecules to regulate group behaviors such as biofilm formation in response to population density. In this study, we attempted to apply the paradigm of bacterial QS to aerobic granular sludge (AGS) formation for wastewater treatment. An essential element of interspecies QS signals, boron, was added to a sequencing batch reactor (SBR) to stimulate AGS growth. Bioassays elaborated the activity of autoinducer-2 (AI-2). We found that boron accelerated AGS growth, resulting in improved settlement performance and increased biomass in the SBR. During continuous SBR operation, the AGS showed an obvious increase in AI-2 activity, which implies that interspecies QS was closely associated with AGS formation. Analysis of EPS showed that boron stimulated tryptophan production, and increased the hydrophobia of AGS. From these results, it was speculated that the addition of boron may have promoted the formation of boron complexed to (R)-4, 5-dihydroxy-2,3-pentanedione (DPD) as the precursor of AI-2, which resulted in accelerated interspecies QS. The results also suggested QS as a novel regulation target for the biogranulation process, such as AGS formation.