Identification of the BolA Protein Reveals a Novel Virulence Factor in K. pneumoniae That Contributes to Survival in Host.

BolA has been characterized as an important transcriptional regulator, which is induced in the stationary phase of growth and is often associated with bacterial virulence. This study was initiated to elucidate the role of the BolA in the virulence of K. pneumoniae. Using a mouse infection model, we revealed bolA mutant strain yielded significantly decreased bacterial loads in the liver, spleen, lung, and kidney, and failed to form liver abscesses. Gene deletion demonstrated that the bolA was required for siderophore production, biofilm formation, and adhesion to human colon cancer epithelial cells HCT116. Quantitative reverse transcriptase PCR (RT-qPCR) indicated that BolA could impact the expression of pulK, pulF, pulE, clpV, vgrG, entE, relA, and spoT genes on a genome-wide scale, which are related to type II secretion system (T2SS), type VI secretion system (T6SS), guanosine tetraphosphate (ppGpp), and siderophore synthesis and contribute to fitness in the host. Furthermore, the metabolome analysis showed that the deletion of the bolA gene led to decreased pools of five metabolites: biotin, spermine, cadaverine, guanosine, and flavin adenine dinucleotide, all of which are involved in pathways related to virulence and stress resistance. Taken together, we provided evidence that BolA was a significant virulence factor in the ability of K. pneumoniae to survive, and this was an important step in progress to an understanding of the pathways underlying bacterial virulence. IMPORTANCE BolA has been characterized as an important transcriptional regulator, which is induced in the stationary phase of growth and affects different pathways directly associated with bacterial virulence. Here, we unraveled the role of BolA in several phenotypes associated with the process of cell morphology, siderophore production, biofilm formation, cell adhesion, tissue colonization, and liver abscess. We also uncovered the importance of BolA for the success of K. pneumoniae infection and provided new clues to the pathogenesis strategies of this organism. This work constitutes a relevant step toward an understanding of the role of BolA protein as a master regulator and virulence factor. Therefore, this study is of great importance for understanding the pathways underlying K. pneumoniae virulence and may contribute to public health care applications.

Carbapenem-resistant K. pneumoniae exhibiting clinically undetected amikacin and meropenem heteroresistance leads to treatment failure in a murine model of infection.

Heteroresistance is a poorly understood mechanism of resistance which refers to a phenomenon where there are different subpopulations of seemingly isogenic bacteria which exhibit a range of susceptibilities to a particular antibiotic. In the current study, we identified a multidrug-resistant, carbapenemase-positive K. pneumoniae strain SWMUF35 which was classified as susceptible to amikacin and resistant to meropenem by clinical diagnostics yet harbored different subpopulations of phenotypically resistant cells, and has the ability to form biofilm. Population analysis profile (PAP) indicated that SWMUF35 showed heteroresistance towards amikacin and meropenem which was considered as co-heteroresistant K. pneumoniae strain. In vitro experiments such as dual PAP, dual Times-killing assays and checkerboard assay showed that antibiotic combination therapy (amikacin combined with meropenem) can effectively combat SWMUF35. Importantly, using an in vivo mouse model of peritonitis, we found that amikacin or meropenem monotherapy was unable to rescue mice infected with SWMUF35. Antibiotic combination therapy could be a rational strategy to use clinically approved antibiotics when monotherapy would fail. Furthermore, our data warn that antibiotic susceptibility testing results may be unreliable due to undetected heteroresistance which can lead to treatment failure and the detection of this phenotype is a prerequisite for a proper choice of antibiotic to support a successful treatment outcome.

Co-infections of two carbapenemase-producing Enterobacter hormaechei clinical strains isolated from the same diabetes individual in China.

Introduction. Since mcr-1 was first reported in China, there have been ten variants of MCR appearing nationwide so far. Multidrug-resistant Enterobacteriaceae bacteria carrying both NDM and MCR have become a serious threat to global public health.Hypothesis/Gap Statement. The genetic structure of mcr-9 needs to be better understood in order to better prevent and control the transmission of drug-resistant genes.Aims. The aim of this study was to characterize the presence of two Enterobacter hormaechei isolates, which carries bla NDM-5 CME2 and the coexistence of mcr-9 and bla NDM-1 strain CMD2, which were isolated from a patient with diabetes in Sichuan, China.Methodology. The microbroth dilution method was used for antibiotic susceptibility. Conjugation experiment was used to investigate the transferability of bla NDM-1, bla NDM-5 and mcr-9. Whole-genome sequencing was performed on Illumina HiSeq platform. The ability of biofilm formation was detected by crystal-violet staining, the virulence of the bacteria was measured by Galleria mellonella killing assay.Results. bla NDM-5 carrier CME2 and CMD2 with bla NDM-1 and mcr-9 were resistant to carbapenems, ß-lactam, aminoglycoside, quinolone and tetracycline, while CMD2 was also resistant to colistin. Conjugation assay and plasmid replicon typing further demonstrated that both bla NDM-1 and bla NDM-5 were respectively present on the self-transferrable IncX3 plasmid, mcr-9 was located on the self-transferrable IncHI2 plasmid. Through the analysis of mcr-9 gene context, the structure was DUF4942-rcnR-rcnA-copS-IS903-mcr-9-wbuC-qseC-qseB-IS1R-ΔsilR-IS903, bla NDM-1 context was IS3000-ΔISAba125-IS5-bla NDM-1-ble-trpF-groS-groL-insE-ΔIS26 structure, bla NDM-5 structure was IS3000-bla NDM-5-ble-trpF-dsbC-ΔIS26-umuD-ISKox3-tnpR-parA. Biofilm formation of CME2 was stronger than CMD2. There was no significant difference in virulence between the two strains.Conclusion. This study reveals two multiple drug-resistant E. hormaechei isolates from diabetes patient samples. E. hormaechei carrying two NDM-resistant genes is already a serious threat, where MCR is an important cause of treatment failure in bacterial infections. This study is a reminder not only to prevent infection in patients with diabetes, but also to constantly monitor the epidemic and spread of the drug-resistant gene.

Heteroresistance to Amikacin in Carbapenem-Resistant Klebsiella pneumoniae Strains.

Heteroresistance can lead to treatment failure and is difficult to detect by the methods currently employed by clinical laboratories. The aim of this study was to investigate the prevalence of the amikacin-heteroresistant Klebsiella pneumoniae strains and explore potential amikacin heteroresistance mechanism through whole-genome sequencing (WGS) and quantitative reverse-transcription PCR (qRT-PCR). In this study, 13 isolates (8.39%) were considered as amikacin-heteroresistant K. pneumoniae strains among a total of 155 K. pneumoniae strains. The majority of the heterogeneous phenotypes (11/13, 84.61%) was unstable and the minimal inhibitory concentrations (MICs) fully or partially reverted back to the level of susceptibility of the parental isolate. The frequency of heteroresistant subpopulation ranged from 2.94×10-7 to 5.59×10-6. Whole-genome sequencing and single-nucleotide variants (SNVs) analysis showed that there were different nucleotide and resultant amino acid alterations among an amikacin-heteroresistant strain S38 and the resistant subpopulation S38L in several genes. Quantitative reverse-transcription PCR analysis revealed that the increased expression of aminoglycoside resistance genes detected in amikacin-heteroresistant K. pneumoniae strains might be associated with amikacin heteroresistance. The findings raise concerns for the emergence of amikacin-heteroresistant K. pneumoniae strains and the use of amikacin as therapy for the treatment of multidrug-resistant K. pneumoniae strains.

Characterization of a novel class 1 integron InSW39 and a novel transposon Tn5393k identified in an imipenem-nonsusceptible Salmonella Typhimurium strain in Sichuan, China.

This study aimed to characterize molecular mechanism of 3 Salmonella enterica strains and novel mobile genetic elements identified in them. The strains, designated SW1, SW39, and SW109084, were obtained from diarrhea patients. The results of susceptibility testing showed SW39 was nonsusceptible to imipenem and cefotaxime. Whole genome sequencing was performed on Illumina HiSeq platform. Multilocus-sequence typing revealed SW1 belonged to ST2529 which was first confirmed in S. enterica, SW109084 was ST34 which was first reported in Enteritidis and SW39 was ST19. Resistome analysis showed SW1, SW109084, and SW39 carried 14, 19, and 17 antibiotic resistance genes. Seven transposons and 4 integrons were confirmed in these strains. Notably, a novel In6- and In7-like class 1 integron designated InSW39 and a novel transposon Tn5393k were identified in plasmid pSW39. The study of genomics and resistance in S. enterica plays a significant role in prevention and treatment of Salmonella infections.