OBJECTIVE: To investigate the relationship between the virulence and the carbapenem resistance phenotype of Klebsiella pneumoniae from blood infection, and to identify carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-HVKP)strains.
METHODS: A total of 192 Klebsiella pneumoniae strains were isolated from blood culture of patients with bloodstream infections from 2016 to 2019, of which 96 isolates were carbapenem-resistant Klebsiella pneumoniae (CRKP) and 96 were carbapenem-sensitive Klebsiella pneumoniae (CSKP). The drug susceptibility was detected by VITEK-2 automatic microbial analyzer; carbapenemase genes, virulence genes and capsule typing were detected by polymerase chain reaction; the high viscosity phenotype of strains was detected by string test, and the genome characteristics of CR-HVKP were detected by whole genome sequencing. Serum killing and biofilm formation test were used to further verify the virulence of CR-HVKP.
RESULTS: There were significant differences in drug resistance to common antibiotics, except for minocycline between CSKP and CRKP isolates (all P<0.05). 92 out of 96 CRKP isolates carried carbapenemase genes, mainly blaKPC-2. The string tests were positive in 4 isolates of CRKP and 36 isolates of CSKP (P<0.05). The detection rates of virulence genes Kfu, aerobictin, iutA, ybtS, rmpA, magA, allS, and capsule antigen K1 and K2 in CSKP group were significantly higher than those in CRKP group (all P<0.05). One HVKP strain was detected in the CRKP group (CR-HVKP) and 36 HVKP was detected in the CSKP group (P<0.05). The CR-HVKP strain belonged to the MLST412, serotype K57, expressed iutA, entB, mrkD, fimH, and rmpA virulence genes, and showed strong biofilm formation and significantly increased serum resistance. Whole genome sequencing results showed that this CR-HVKP isolate carried blaSHV-145, blaTEM-1, blaCTX-M-3, fosA6, oqxA5, oqxB26, and aac(3)-IId resistance genes, accompanied by abnormalities in outer membrane protein K (OmpK) 35 and OmpK36.
CONCLUSIONS: The drug resistance of CRKP is significantly higher than that of CSKP, while CRKP carrying fewer virulence genes in both number and types compared to CSKP. A new MLST type of carbapenem-resistant and hypervirulent Klebsiella pneumoniae strain has been detected, which requires clinical awareness and epidemiological monitoring.
目的: 探讨本地区从血培养分离的肺炎克雷伯菌临床株的毒力与碳青霉烯类耐药表型的关系，并对碳青霉烯类耐药高毒力肺炎克雷伯菌（CR-HVKP）进行毒力表型验证。方法: 收集2016—2019年血流感染患者血培养分离的192株非重复肺炎克雷伯菌，其中96株为碳青霉烯类耐药肺炎克雷伯菌（CRKP），96株为碳青霉烯敏感肺炎克雷伯菌（CSKP）。采用VITEK-2全自动微生物分析仪检测菌株对药物的敏感性，聚合酶链反应检测碳青霉烯类耐药基因、毒力基因和荚膜分型，拉丝实验检测菌株的高黏表型，全基因组测序方法检测CR-HVKP菌株的基因组特征，并通过血清抗性试验和生物膜形成试验进一步验证CR-HVKP的毒力。结果: CRKP对常见抗菌药物耐药率高，CSKP则对常见抗菌药物较敏感；除米诺环素外，CRKP组和CSKP组菌株对抗菌药物的耐药率差异均有统计学意义（均P<0.05）。96株CRKP中，92株携带碳青霉烯酶基因，以blaKPC-2为主。在毒力因子方面，4株CRKP和36株CSKP拉丝试验阳性，呈现为高黏表型，两者差异具有统计学意义（P<0.05）。与CRKP组比较，CSKP组Kfu、aerobictin、iutA、ybtS、rmpA、magA、allS等毒力基因检出率均增加，荚膜抗原K1和K2检出率也增加（均P<0.05）。CRKP组检出高毒力肺炎克雷伯菌（HVKP）1株（即CR-HVKP），CSKP组检出HVKP 36株，差异有统计学意义（P<0.05）。全基因组测序结果显示，CR-HVKP多位点序列分型为412，荚膜抗原为K57，携带iutA、entB、mrkD、fimH和rmpA毒力基因，生物膜形成能力和血清抵抗力强，同时携带blaSHV-145、blaTEM-1、blaCTX-M-3、fosA6、oqxA5、oqxB26和aac（3）-IId耐药基因，伴随外膜蛋白K（OmpK）35和OmpK36的异常。结论: CRKP对抗菌药物的耐药率明显高于CSKP，虽然其携带的毒力基因数和种类均少于CSKP，但也出现了碳青霉烯类耐药且高毒力的新型多位点序列分型肺炎克雷伯菌。后者可能对公共卫生造成严重威胁。.

Cefiderocol, a siderophore-cephalosporine conjugate antibiotic, shows promise as a therapeutic option for carbapenem-resistant (CR) Acinetobacter infections. While resistance has already been reported in A. baumannii, combination therapies with avibactam or sulbactam reduce MICs of cefiderocol, extending its efficacy. However, careful consideration is necessary when using these combinations. In our experiments, exposure of A. baumannii and A. lwoffii to cefiderocol and sulbactam or avibactam led to the selection of cefiderocol-resistant strains. Three of those were subjected to whole genome sequencing and transcriptomic analysis. The strains all possessed synonymous and non-synonymous substitutions and short deletions. The most significant mutations affected efflux pumps, transcriptional regulators, and iron homeostasis genes. Transcriptomics showed significant alterations in expression levels of outer membrane proteins, iron homeostasis, and β-lactamases, suggesting adaptive responses to selective pressure. This study underscores the importance of carefully assessing drug synergies, as they may inadvertently foster the selection of resistant variants and complicate the management of CR Acinetobacter infections.IMPORTANCEThe emergence of carbapenem-resistant Acinetobacter strains as a serious global health threat underscores the urgent need for effective treatment options. Although few drugs show promise against CR Acinetobacter infections, resistance to both drugs has been reported. In this study, the molecular characterization of spontaneous cefiderocol-resistant variants, a CR A. baumannii strain with antagonism to sulbactam, and an A. lwoffii strain with antagonism to avibactam, provides valuable insights into the mechanisms of resistance to cefiderocol. Some mechanisms observed are associated with mutations affecting efflux pumps, regulators, and iron homeostasis genes. These findings highlight the importance of understanding resistance mechanisms to optimize treatment options. They also emphasize the importance of early evaluation of drug synergies to address the challenges of antimicrobial resistance in Acinetobacter infections.

Carbapenem-resistant Acinetobacter baumannii (CRAB) is a major human pathogen and a research priority for developing new antimicrobial agents. CRAB is a causative agent of a variety of infections in different body sites. One of the manifestations is catheter-associated urinary tract infection, which exposes the bacteria to the host\'s urine, creating a particular environment. Exposure of two CRAB clinical isolates, AB5075 and AMA40, to human urine (HU) resulted in the differential expression levels of 264 and 455 genes, respectively, of which 112 were common to both strains. Genes within this group play roles in metabolic pathways such as phenylacetic acid (PAA) catabolism, the Hut system, the tricarboxylic acid (TCA) cycle, and other processes like quorum sensing and biofilm formation. These results indicate that the presence of HU induces numerous adaptive changes in gene expression of the infecting bacteria. These changes presumably help bacteria establish and thrive in the hostile conditions in the urinary tract. These analyses advance our understanding of CRAB\'s metabolic adaptations to human fluids, as well as expand knowledge on bacterial responses to distinct human fluids containing different concentrations of human serum albumin (HSA).

The aim of this study is to evaluate the antimicrobial susceptibility of invasive isolates of Serratia marcescens, associated with blood stream infections (BSIs) in patients hospitalized in Varna University Hospital, Bulgaria, as well as to identify the genetic mechanisms responsible for 3rd generation cephalosporin and carbapenem-resistance among these isolates. A total of 45 consecutive S. marcescens isolates, obtained from blood cultures of 45 patients with BSIs, hospitalized during an 8-year period (2016-2023) were included. Species identification and antimicrobial susceptibility testing were done by Phoenix (BD, USA) and Vitek 2 (BioMerieux, France) systems and the results were interpreted according to EUCAST guidelines. The genetic mechanisms of beta-lactam resistance were studied by PCR. During the study period, a total of 45 patients were diagnosed with S. marcescens-associated BSIs. All infections were defined as nosocomial, predominantly intensive care unit-acquired (42.2%) and 28.8% were central venous catheter-associated. The following antimicrobial resistance rates were found: ceftriaxone, piperacillin/tazobactam, 57.8%; ceftazidime, 55.6%; cefepime, trimethoprime/sulfamethoxazole, 53.3%; gentamicin, 48.8%; ciprofloxacin, 44.5%; amikacin, 15.6%; carbapenems, 2.2%. The blaCTX-M was identified in 88.9% of the tested 3rd generation cephalosporin resistant isolates. Among these, 50% were also blaTEM positive. The single carbapenem-resistant isolate harboured blaKPC, blaCTX-M1/9, blaCMY-2 and blaTEM. This study demonstrates S. marcescens as a problematic nosocomial pathogen and we report a KPC-producing S. marcescens clinical isolate from a BSI in Bulgaria.

Carbapenem-resistant Klebsiella pneumoniae (CPKP) infections seriously threaten global public health. The main objective of this study was to assess the in-vitro synergistic activity of ceftazidime-avibactam (CZA) in combination with colistin (COL), amikacin (AK), gentamicin (GEN), and fosfomycin (FOS) against CPKP isolates. The secondary goal was to determine the antibiotic susceptibility performance of BD Phoenix. OXA-48 (49.1%) was the predominant carbapenemase, followed by KPC (29.1%). We used the broth microdilution (BMD) method to determine the minimum inhibitory concentrations (MICs) of CZA, COL, AK, and GEN. Meanwhile, the MICs of FOS were determined by the agar dilution (AD) method. To examine the antibacterial activity of CZA, we conducted a checkerboard assay (CBA) with COL, AK, GEN, and FOS against CRKP isolates. We randomly selected three strains and performed synergy testing via time-kill assay (TKA). CRKP isolates were 89.1% susceptible to CZA, 16.4% to COL, 21.8% to GEN, and 29.1% to AK using BMD, 47.3% to FOS by AD. The most synergistic effects were observed in the combination of CZA-COL (78.2%) and CZA-FOS (63.6%). Given the limited therapeutic options for treating severe CRKP infections, combining CZA with COL and FOS may enhance in-vitro activity against clinical CRKP isolates.

Public health faces daily challenges due to increasing reports of pathogenic microorganisms with new antimicrobial resistance. Klebsiella michiganensis, an emerging pathogen, poses difficulty in its identification using conventional techniques. This study presents the first documented case of NDM-1-producing K. michiganensis in Brazil, identified as the new ST418. Initially, the isolate from a tracheal secretion was misidentified as K. oxytoca. However, accurate identification was achieved through ANI analyses. Whole-genome sequencing was conducted to characterize the genetic context of the resistance genes, to identify virulence factors, and to construct a phylogenetic tree. The blaNDM-1 gene was found to be harbored on an IncFIB plasmid approximately 112 kb in length, which was transferable in conjugation assays. The detection of carbapenem resistance genes in this species highlights the importance of public health vigilance, as it may serve as a reservoir and disseminator of significant resistance genes.

Carbapenem-resistant Enterobacteriaceae (CRE) have diminished treatment options causing serious morbidities and mortalities. This systematic review and meta-analysis assessed the prevalence and associated factors of Enterobacteriaceae infections in clinical, livestock and environmental settings globally. The population intervention comparison and outcome strategy was used to enroll studies using the preferred reporting system for systematic review and meta-analysis to include only cross-sectional studies. Search engines used to retrieve articles included journal author name estimator, PubMed, Google Scholar and African Journals Online (AJOL). The Newcastle-Ottawa scale was used to assess the quality of studies. Sixteen articles from 2013 to 2023 in Africa, Asia, Europe and South America were studied. The pooled prevalence of CRE was 43.06% (95% CI 21.57-66.03). Klebsiella pneumoniae (49.40%), Escherichia coli (26.42%), and Enterobacter cloacae (14.24%) were predominant. Klebsiella pneumoniae had the highest resistance with the blaKPC-2 in addition to blaNDM, blaOXA-48, blaIMP and blaVIM. The blaKPC-2 genes occurrence was associated with environmental (P-value < 0.0001) and South American studies (P-value < 0.0001), but there was no difference in the trends over time (P-value = 0.745). This study highlights the high rates of CRE infections, particularly within blaKPC production. Monitoring and surveillance programs, research and infection control measures should be strengthened. Additionally, further studies are needed to explore the mechanisms driving the predominance of specific bacterial species and the distribution of resistance genes within this bacterial family.

Antimicrobial resistance has become a growing public health threat in recent years. Klebsiella pneumoniae is one of the priority pathogens listed by the World Health Organization. Antimicrobial peptides are considered promising alternatives to antibiotics due to their broad-spectrum antibacterial activity and low resistance. In this study, we investigated the antibacterial activity of antimicrobial peptide A20L against K. pneumoniae. In vitro antibacterial activity of A20L against K. pneumoniae was demonstrated by broth microdilution method. We confirmed the in vivo efficacy of A20L by Galleria mellonella infection model. In addition, we found that A20L also had certain antibiofilm activity by crystal violet staining. We also evaluated the safety and stability of A20L, and the results revealed that at a concentration of ≤128 µg/mL, A20L exhibited negligible toxicity to RAW264.7 cells and no substantial toxicity to G. mellonella. A20L was stable at different temperatures and with low concentration of serum [5% fetal bovine serum (FBS)]; however, Ca2+, Mg2+, and high serum concentrations reduced the antibacterial activity of A20L. Scanning electron microscope (SEM) and membrane permeability tests revealed that A20L may exhibit antibacterial action by damaging bacterial cell membranes and increasing the permeability of outer membrane. Taken together, our results suggest that A20L has significant development potential as a therapeutic antibiotic alternative, which provides ideas for the treatment of K. pneumoniae infection.
OBJECTIVE: A20L showed antibacterial and anti-infective efficacy in vitro and in vivo against Klebsiella pneumoniae. It can have an antibacterial effect by disrupting the integrity of cell membranes. A20L displayed anti-biofilm and anti-inflammatory activity against carbapenem-resistant K. pneumoniae and certain application potential in vivo, which provides a new idea for the clinical treatment of biofilm-associated infections.

Recently, considerable uncertainty has arisen concerning the appropriate susceptibility testing for cefiderocol in gram-negative bacilli, particularly in the context of its application to Acinetobacter spp. The optimal method for assessing the susceptibility levels of Acinetobacter spp. to cefiderocol remains a subject of debate due to substantial disparities observed in the values obtained through various testing procedures. This study employed four minimum inhibitory concentration (MIC) methodologies and the disk diffusion to assess the susceptibility of twenty-seven carbapenem resistant (CR)-Acinetobacter strains to cefiderocol. The results from our study reveal significant variations in the minimum inhibitory concentration (MIC) values obtained with the different methods and in the level of agreement in interpretation categories between the different MIC methods and the disk diffusion test. Among the MIC methods, there was relatively more consistency in reporting the interpretation categories. For European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints, the categorical agreement (CA) for MIC methods ranged between 66.7 and 81.5%. On the other hand, the essential agreement (EA) values were as low as 18.5-29.6%. The CA between MIC methods and disk diffusion was 81.5%. These results emphasize the need for a reliable, accurate, and clinically validated methodology to effectively assess the susceptibility of Acinetobacter spp. to cefiderocol. The wide variability observed in our study highlights the importance of standardizing the susceptibility testing process for cefiderocol to ensure consistent and reliable results for clinical decision-making.

Carbapenem-resistant Acinetobacter baumannii (CRAB) is a major human pathogen and a research priority for developing new antimicrobial agents. CRAB is a causative agent of a variety of infections in different body sites. One of the manifestations is catheter-associated urinary tract infection, which exposes the bacteria to the host\'s urine, creating a particular environment. Exposure of two CRAB clinical isolates, AB5075 and AMA40, to human urine (HU) resulted in the differential expression levels of 264 and 455 genes, respectively, of which 112 were common to both strains. Genes within this group play roles in metabolic pathways such as phenylacetic acid (PAA) catabolism, the Hut system, the tricarboxylic acid (TCA) cycle, and other processes like quorum sensing and biofilm formation. These results indicate that the presence of HU induces numerous adaptive changes in gene expression of the infecting bacteria. These modifications presumably help bacteria establish and thrive in the hostile conditions in the urinary tract. These analyses advance our understanding of CRAB\'s metabolic adaptations to human fluids, as well as expanding knowledge on bacterial responses to distinct human fluids containing different concentrations of human serum albumin (HSA).