Background: Klebsiella pneumoniae is considered the most clinically relevant species of Enterobacteriaceae, known to cause severe infections including liver abscesses. To the best of our knowledge, a large proportion of iron in the human body is accumulated and stored in the liver. We hypothesize that increased iron availability is an important factor driving liver abscess formation and we therefore aim to understand the effects of iron on K. pneumoniae causing liver abscesses. Results: All tested K. pneumoniae clinical isolates, including those isolated from liver abscesses and other abdominal invasive infection sites, grew optimally when cultured in LB broth supplemented with 50 μM iron and exhibited the strongest biofilm formation ability under those conditions. Decreased growth and biofilm formation ability were observed in all tested strains when cultured with an iron chelator (P < 0.05). The infection model of G. mellonella larvae indicated the virulence of liver abscess-causing K. pneumoniae (2/3) cultured in LB broth with additional iron was significantly higher than those under iron-restricted conditions (P < 0.05). The relative expression levels of the four siderophore genes (iucB, iroB, irp1, entB) in K. pneumoniae strains isolated from liver abscesses cultured with additional iron were lower than those under iron-restricted conditions (P < 0.05). Conclusions: It is suggested by our research that iron in the environment can promote growth, biofilm formation and enhance virulence of K. pneumoniae causing liver abscesses. A lower expression of siderophore genes correlates with increased virulence of liver abscess-causing K. pneumoniae. Further deeper evaluation of these phenomena is warranted.

Background: OXA-232-producing carbapenem-resistant Klebsiella pneumoniae (CRKP) has the potential to become the “third epidemic” of carbapenem-resistant Klebsiella strain after KPC-2 and NDM in China. We investigated the first outbreak of CRKP in the First Affiliated Hospital of Wenzhou Medical University. Methods: We collected 610 clinical isolates of CRKP from the First Affiliated Hospital of Wenzhou Medical University between January 2019 and September 2020 and screened them by Polymerase Chain Reaction (PCR). The multilocus sequence typing and pulsed-field gel electrophoresis were used to determine the genetic relatedness of the strains. The antimicrobial susceptibility test was performed to determine the drug resistance of the clinical isolates. The molecular mechanism underlying carbapenem resistance was elucidated by performing PCR and conjugation experiments. The virulence potential of the strains was determined by the string test, detection of virulence-associated genes and capsular serotypes, and Galleria mellonella larval infection model. Results: Between September 2019 and May 2020, 26 OXA-232-producing CRKP were obtained from 12 patients in our hospital. Ten patients were hospitalized in the intensive care units (ICU) and the overall mortality of the inpatients involved in the outbreak was 50% (6/12). Epidemiological investigations reported that all the OXA-232-producing CRKP strains belonged to the sequence type ST15 and can be clonally transmitted among the inpatients in the ICU. All the strains had low virulence and were resistant to commonly used clinical antibiotics except for ceftazidime/avibactam, colistin, and tigecycline. The OXA-232-producing CRKP was sensitive to triclosan and chlorhexidine, and its eradication from our hospital can be achieved by the use of disinfectants in the ICU. Conclusion: In our study, OXA-232-producing CRKP isolates appeared to be clonally transmitted and the sequence type ST15 was responsible for the outbreak. Therefore, effective measurements for the infection control of CRKP are urgently needed to prevent its epidemic in the nearby region in the future.

During March 2017, a neonatal patient with severe diarrhoea subsequently developed septicaemia and died, with Klebsiella isolated as the causative microorganism. In keeping with infection control protocols, the coincident illness of an attending staff member and three other neonates with Klebsiella infection triggered an outbreak response, leading to microbiological assessment of isolates collected from the staff member and all 21 co-housed neonates. Multilocus sequence typing and genomic sequencing identified that the isolates from the 21 neonates were of a new Klebsiella sequence type, ST2727, and taxonomically belonged to K. quasipneumoniae subsp. similipneumoniae (formerly referred to as KpIIB). Genomic characterization showed that the isolated ST2727 strains had diverged from other K. quasipneumoniae subsp. similipneumoniae strains at least 90 years ago, whereas the neonatal samples were highly similar with a genomic divergence of 3.6 months. There was no relationship to the Klebsiella isolate from the staff member. This demonstrates that no transmission occurred from staff to patient or between patients. Rather, the data suggest that ST2727 colonized each neonate from a common hospital source. Sequence-based analysis of the genomes revealed several genes for antimicrobial resistance and some virulence features, but suggest that ST2727 is neither extremely-drug resistant nor hypervirulent. Our results highlight the clinical significance and genomic properties of ST2727 and urge genome-based measures be implemented for diagnostics and surveillance within hospital environments. Additionally, the present study demonstrates the need to scale the power of genomic analysis in retrospective studies where relatively few samples are available.

Colistin is a polymyxin antibiotic that is widely used for the treatment of multidrug resistant (MDR) Pseudomonas aeruginosa infections, as the last resort. Over the past few years, unreasonable use of antibiotics has resulted in an increase in MDR strains, including colistin-resistant P. aeruginosa. The present study aimed to explore the synergistic effects of mefloquine in combination with colistin for the treatment of colistin-resistant P. aeruginosa in vivo and in vitro. The synergistic effect of the combination of mefloquine and colistin was investigated in vitro using checkerboard method, time-killing assay, biofilm formation inhibition test, and biofilm eradication test. The study also explored the synergistic effects of this combination of drugs in vivo, using a Galleria mellonella infection model. The results for checkerboard method and time killing curve indicated that mefloquine in combination with colistin showed a good antibacterial activity. Furthermore, the combination of these two drugs inhibited biofilm formation and eradicated pre-formed mature biofilms. This synergistic effect was visualized using scanning electron microscopy (SEM), wherein the results showed that the combination of mefloquine and colistin reduced biofilm formation significantly. Further, the application of this combination of drugs to in vivo infection model significantly increased the survival rate of G. mellonella larvae. Altogether, the combination of mefloquine and colistin showed a good synergistic effect in vitro and in vivo, and highlighted its potential to be used as an alternative therapy for the treatment of colistin-resistant P. aeruginosa infection.

The bacteriophage is an effective adjunct to existing antibiotic therapy; however, in the course of bacteriophage therapy, host bacteria will develop resistance to bacteriophages, thus affecting the efficacy. Therefore, it is important to describe how bacteria evade bacteriophage attack and the consequences of the biological changes that accompany the development of bacteriophage resistance before the bacteriophage is applied. The specific bacteriophage vB3530 of Pseudomonas aeruginosa ( P. aeruginosa ) has stable biological characteristics, short incubation period, strong in vitro cleavage ability, and absence of virulence or resistance genes. Ten bacteriophage-resistant strains (TL3780-R) were induced using the secondary infection approach, and the plaque assay showed that vB3530 was less sensitive to TL3780-R. Identification of bacteriophage adsorption receptors showed that the bacterial surface polysaccharide was probably the adsorption receptor of vB3530. In contrast to the TL3780 parental strain, TL3780-R is characterized by the absence of long lipopolysaccharide chains, which may be caused by base insertion of wzy or deletion of galU . It is also intriguing to observe that, in comparison to the parent strain, the bacteriophage-resistant strains TL3780-R mostly exhibited a large cost of fitness (growth rate, biofilm formation, motility, and ability to produce enhanced pyocyanin). In addition, TL3780-R9 showed increased susceptibility to aminoglycosides and chlorhexidine, which may be connected to the loss and down-regulation of mexX expression. Consequently, these findings fully depicted the resistance mechanism of P. aeruginosa to vB3530 and the fitness cost of bacteriophage resistance, laying a foundation for further application of bacteriophage therapy. IMPORTANCE: The bacteriophage is an effective adjunct to existing antibiotic therapy; However, bacteria also develop defensive mechanisms against bacteriophage attack. Thus, there is an urgent need to deeply understand the resistance mechanism of bacteria to bacteriophages and the fitness cost of bacteriophage resistance so as to lay the foundation for subsequent application of the phage. In this study, a specific bacteriophage vB3530 of P. aeruginosa had stable biological characteristics, short incubation period, strong in vitro cleavage ability, and absence of virulence or resistance genes. In addition, we found that P. aeruginosa may lead to phage resistance due to the deletion of galU and the base insertion of wzy , involved in the synthesis of lipopolysaccharides. Simultaneously, we showed the association with the biological state of the bacteria after bacteria acquire bacteriophage resistance, which is extremely relevant to guide the future application of therapeutic bacteriophages.

This study aimed to elucidate resistance to carbapenems and fluoroquinolones, the transmission mechanism of bla_KPC-2, and the virulence characteristics of a Pseudomonas aeruginosa strain (TL3773) isolated in East China. The virulence and resistance mechanisms of TL3773 were investigated by whole genome sequencing (WGS), comparative genomic analysis, conjugation experiments, and virulence assays. This study isolated carbapenem-resistant P. aeruginosa from blood resistant to carbapenems. The patient's clinical data showed poor prognosis compounded by multiple sites of infection. WGS showed that TL3773 carried aph (3')-IIb, bla_PAO, bla_OXA-486, fosA, catB7, and two crpP resistance genes on chromosome, and the carbapenem resistance gene bla_KPC-2 on plasmid. We identified a novel crpP gene named TL3773-crpP2. Cloning experiments proved that TL3773-crpP2 was not the primary cause of fluoroquinolone resistance in TL3773. GyrA and ParC mutations may confer fluoroquinolone resistance. The bla_KPC-2 genetic environment was IS26-TnpR-ISKpn27-bla_KPC-2-ISKpn6-IS26-Tn3-IS26, potentially mediating the transmission of bla_KPC-2 in P. aeruginosa. The overall virulence of TL3773 was lower than that of PAO1. However, the pyocyanin and biofilm formation of TL3773 was higher than that of PAO1. WGS further indicated that TL3773 was less virulent than PAO1. Phylogenetic analysis showed that TL3773 was most similar to the P. aeruginosa isolate ZYPA29 from Hangzhou, China. These observations further indicate that ST463 P. aeruginosa is rapidly spreading. The threat of ST463 P. aeruginosa harbouring bla_KPC-2 is emergent and may pose a threat to human health. More extensive surveillance and effective actions are urgently needed to control its further spread.