Genotypic-phenotypic discordance for cefiderocol against Enterobacter hormaechei.

Mobile colistin resistance (mcr) genes represent an emerging threat to public health. Reports on the prevalence, antimicrobial profiles, and clonality of MCR-9-producing Enterobacter cloacae complex (ECC) isolates on a national scale in China are limited. We screened 3,373 samples from humans, animals, and the environment and identified eleven MCR-9-positive ECC isolates. We further investigated their susceptibility, epidemiology, plasmid profiles, genetic features, and virulence potential. Ten strains were isolated from severe bloodstream infection cases, especially three of them were recovered from neonatal sepsis. Enterobacter hormaechei was the most predominant species among the MCR-9-producing ECC population. Moreover, the co-existence of MCR-9, CTX-M, and SHV-12 encoding genes in MCR-9-positive isolates was globally observed. Notably, mcr-9 was mainly carried by IncHI2 plasmids, and we found a novel ~187 kb IncFII plasmid harboring mcr-9, with low similarity with known plasmids. In summary, our study presented genomic insights into genetic characteristics of MCR-9-producing ECC isolates retrieved from human, animal, and environment samples with one health perspective. This study is the first to reveal NDM-1- and MCR-9-co-producing ECC from neonatal sepsis in China. Our data highlights the risk for the hidden spread of the mcr-9 colistin resistance gene.

This study investigated the clinical and genomic characteristics of Enterobacter cloacae complex (ECC) and Klebsiella aerogenes bloodstream infections (BSIs) in pediatric patients. A total of 115 BSI episodes (ECC: 86, K. aerogenes: 29) from 110 children hospitalized between 2011 and 2024 were retrospectively analyzed. Whole-genome sequencing was performed on available isolates to determine species, sequence types, and antimicrobial resistance (AMR) genes. Clinical characteristics, antibiotic usage, and outcomes were compared between groups. Patients with K. aerogenes BSI were younger and more likely to be preterm or diagnosed with urosepsis, while ECC infections were more frequently associated with hematologic malignancies. According to a multivariable analysis of the entire cohort (n = 115), K. aerogenes infection (OR [6.26], 95% CI [1.36-28.78]) and gentamicin resistance (OR [10.06], 95% CI [1.88-53.87]) were independently associated with 30-day mortality. Enterobacter hormaechei was the most common ECC species (68.4%) and exhibited the highest prevalence of AMR genes, particularly those conferring resistance to aminoglycosides, β-lactams, and trimethoprim-sulfamethoxazole. In contrast, K. aerogenes harbored few resistance genes. Multi-locus sequence typing analysis revealed high genetic diversity in both ECC and K. aerogenes, without evidence of dominant clonal expansion. Despite similarities in clinical presentation, ECC and K. aerogenes exhibit distinct age distributions, resistance profiles, and genetic diversity in pediatric BSIs. These findings underscore the importance of species-level identification and continued genomic surveillance to inform empirical antibiotic strategies and prevent the spread of resistant strains.

Enterobacter cloacae complex (ECC) is composed of multiple species and the taxonomic status is consecutively updated. In last decades ECC is frequently associated with multidrug resistance and become an important nosocomial pathogen. Currently, rapid and accurate identification of ECC to the species level remains a technical challenge, thus impedes our understanding of the population at the species level. Here, we aimed to develop a simple, reliable, and economical method to distinguish four epidemiologically prevalent species of ECC with clinical significance, i.e., E. cloacae, E. hormaechei, E. roggenkampii, and E. kobei. A total of 977 ECC genomes were retrieved from the GenBank, and unique gene for each species was obtained by core-genome comparisons. Four pairs of species-specific primers were designed based on the unique genes. A total of 231 ECC clinical strains were typed both by hsp60 typing and by species-specific PCRs. The specificity and sensitivity of the four species-specific PCRs ranged between 96.56% and 100% and between 76.47% and 100%, respectively. The PCR for E. cloacae showed the highest specificity and sensitivity. A one-step multiplex PCR was subsequently established by combining the species-specific primers. Additional 53 hsp60-typed ECC and 20 non-ECC isolates belonging to six species obtained from samples of patients, sewage water and feces of feeding animals were tested by the multiplex PCR. The identification results of both techniques were concordant. The multiplex PCR established in this study provides an accurate, expeditious, and cost-effective way for routine diagnosis and molecular surveillance of ECC strains at species level.

Multidrug-resistant strains belonging to the Enterobacter cloacae complex (ECC) group, and especially those belonging to clusters C-III, C-IV, and C-VIII, have increasingly emerged as a leading cause of health care-associated infections, with colistin used as one of the last lines of treatment. However, colistin-resistant ECC strains have emerged. The aim of this study was to prove that MgrB, the negative regulator of the PhoP/PhoQ two-component regulatory system, is involved in colistin resistance in ECC of cluster C-VIII, formerly referred to as Enterobacter hormaechei subsp. steigerwaltii An in vitro mutant (Eh22-Mut) was selected from a clinical isolate of Eh22. The sequencing analysis of its mgrB gene showed the presence of one nucleotide deletion leading to the formation of a truncated protein of six instead of 47 amino acids. The wild-type mgrB gene from Eh22 and that of a clinical strain of Klebsiella pneumoniae used as controls were cloned, and the corresponding recombinant plasmids were used for complementation assays. The results showed a fully restored susceptibility to colistin and confirmed for the first time that mgrB gene expression plays a key role in acquired resistance to colistin in ECC strains.

Genomic analysis of IMP-8-producing Enterobacter hormaechei with a novel plasmid pK432-IMP

ABSTRACTMultidrug resistance (MDR) in Enterobacteriaceae including resistance to quinolones is rising worldwide. The plasmid-mediated quinolone resistance (PMQR) gene qnrS is prevalent in Enterobacteriaceae. However, the qnrS gene is rarely found in Enterobacter hormaechei (E. hormaechei). Here, we reported one multidrug resistant E. hormaechei strain M1 carrying the qnrS1 and blaTEM-1 genes. This study was to analyze the characteristics of MDR E. hormaechei strain M1. The E. hormaechei strain M1 was identified as Enterobacter cloacae complex by biochemical assay and 16S rRNA sequencing. The whole genome was sequenced by the Oxford Nanopore method. Taxonomy of the E. hormaechei was based on multilocus sequence typing (MLST). The qnrS with the other antibiotic resistance genes were coexisted on IncF plasmid (pM1). Besides, the virulence factors associated with pathogenicity were also located on pM1. The qnrS1 gene was located between insertion element IS2A (upstream) and transposition element ISKra4 (downstream). The comparison result of IncF plasmids revealed that they had a common plasmid backbone. Susceptibility experiment revealed that the E. hormaechei M1 showed extensive resistance to the clinical antimicrobials. The conjugation transfer was performed by filter membrane incubation method. The competition and plasmid stability assays suggested the host bacteria carrying qnrS had an energy burden. As far as we know, this is the first report that E. hormaechei carrying qnrS was isolated from chicken feed. The chicken feed and poultry products could serve as a vehicle for these MDR bacteria, which could transfer between animals and humans through the food chain. We need to pay close attention to the epidemiology of E. hormaechei and prevent their further dissemination.IMPORTANCE Enterobacter hormaechei is an opportunistic pathogen. It can cause infections in humans and animals. Plasmid-mediated quinolone resistance (PMQR) gene qnrS can be transferred intergenus, which is leading to increase the quinolone resistance levels in Enterobacteriaceae. Chicken feed could serve as a vehicle for the MDR E. hormaechei. Therefore, antibiotic-resistance genes (ARGs) might be transferred to the intestinal flora after entering the gastrointestinal tract with the feed. Furthermore, antibiotic-resistant bacteria (ARB) were also excreted into environment with feces, posing a huge threat to public health. This requires us to monitor the ARB and antibiotic-resistant plasmids in the feed. Here, we demonstrated the characteristics of one MDR E. hormaechei isolate from chicken feed. The plasmid carrying the qnrS gene is a conjugative plasmid with transferability. The presence of plasmid carrying antibiotic-resistance genes requires the maintenance of antibiotic pressure. In addition, the E. hormaechei M1 belonged to new sequence type (ST). These data show the MDR E. hormaechei M1 is a novel strain that requires our further research.

Enterobacter hormaechei has emerged as a significant pathogen within healthcare settings due to its ability to develop multidrug resistance (MDR) and survive in hospital environments. This study presents a genome-based analysis of carbapenem-resistant Enterobacter hormaechei isolates from two major hospitals in the United Arab Emirates. Eight isolates were subjected to whole-genome sequencing (WGS), revealing extensive resistance profiles including the blaNDM-1, blaOXA-48, and blaVIM-4 genes. Notably, one isolate belonging to ST171 harbored dual carbapenemase genes, while five isolates exhibited colistin resistance without mcr genes. The presence of the type VI secretion system (T6SS), various adhesins, and virulence genes contributes to the virulence and competitive advantage of the pathogen. Additionally, our isolates (87.5%) possessed ampC β-lactamase genes, predominantly blaACT genes. The genomic context of blaNDM-1, surrounded by other resistance genes and mobile genetic elements, highlights the role of horizontal gene transfer (HGT) in the spread of resistance. Our findings highlight the need for rigorous surveillance, strategic antibiotic stewardship, and hospital-based WGS to manage and mitigate the spread of these highly resistant and virulent pathogens. Accurate identification and monitoring of Enterobacter cloacae complex (ECC) species and their resistance mechanisms are crucial for effective infection control and treatment strategies.

Colistin has emerged as the last resort for treating multidrug-resistant Enterobacter cloacae complex (ECC) infections. The primary purposes of this study were to demonstrate the presence of colistin heteroresistance in ECC and to further investigate their clinical characteristics, molecular epidemiology and mechanisms. Population analysis profiles (PAP) were performed to confirm the heteroresistance phenotype. Average nucleotide identity (ANI) was determined to classify ECC species. Phylogenetic analysis based on core genome single nucleotide polymorphisms (cg-SNPs), multilocus sequence typing (MLST) and core genome MLST (cg-MLST). Risk factors and clinical outcomes of infections were analyzed through a retrospective case-control study. Potential mechanisms of colistin heteroresistance were evaluated using polymerase chain reaction (PCR), efflux pump inhibition assays and reverse transcription quantitative PCR (RT-qPCR). A high proportion (24.4%) of the non-resistant strains were colistin-heteroresistant isolates. Among the several ECC species, Enterobacter kobei had the largest percentage (29.4%) of colistin-heteroresistant isolates, followed by Enterobacter hormaechei (20.5%) and Enterobacter bugandensis (20.0%). Notably, only one strain (0.8%; 1/132) of Enterobacter hormaechei was fully resistant to colistin. Different ECC species showed varying heteroresistance levels: Enterobacter roggenkampii, Enterobacter kobei, Enterobacter asburiae and Enterobacter bugandensis displayed high heteroresistancelevels(MIC ≥ 128 mg/L). 75% of all ST116 and ST56 strains were heteroresistant to colistin. The infection of ST116 and ST56 strains as well as exposure to cephalosporin antibiotics were independent risk factors for colistin-heteroresistant ECC infections. Mechanistic analysis revealed that heteroresistance strongly correlated with the overexpression of arnA, regulated by the PhoPQ two-component system (TCS). Notably, mgrB had minimal impact. AcrAB-TolC efflux pump genes showed unsynchronized expression; High acrB expression was strongly associated with colistin heteroresistance, while acrA and tolC were not. Colistin heteroresistance showed species-dependent variations in levels and prevalence rates. The colistin-heteroresistant mechanisms were complex, involving coordinated regulation of multiple genes. These results highlighted the need for tailored antimicrobial stewardship. In addition, the development of direct, reliable and rapid clinical methods for detecting heteroresistance is essential for improving infection management and prevention.

New sequence type of an Enterobacter cloacae complex strain with the potential to become a high-risk clone

According to the World Health Organization, carbapenem-resistant Enterobacteriaceae (CRE) belong to the highest priority group for the development of new antibiotics. Argentina-WHONET data showed that Gram-negative resistance frequencies to imipenem have been increasing since 2010 mostly in two CRE bacteria: Klebsiella pneumoniae and Enterobacter cloacae Complex (ECC). This scenario is mirrored in our hospital. It is known that K. pneumoniae and the ECC coexist in the human body, but little is known about the outcome of these species producing KPC, and colonizing or infecting a patient. We aimed to contribute to the understanding of the rise of the ECC in Argentina, taking as a biological model both a patient colonized with two KPC-producing strains (one Enterobacter hormaechei and one K. pneumoniae) and in vitro competition assays with prevalent KPC-producing ECC (KPC-ECC) versus KPC-producing K. pneumoniae (KPC-Kp) high-risk clones from our institution. A KPC-producing E. hormaechei and later a KPC-Kp strain that colonized a patient shared an identical novel conjugative IncM1 plasmid harboring bla KPC-2. In addition, a total of 19 KPC-ECC and 58 KPC-Kp strains isolated from nosocomial infections revealed that high-risk clones KPC-ECC ST66 and ST78 as well as KPC-Kp ST11 and ST258 were prevalent and selected for competition assays. The competition assays with KCP-ECC ST45, ST66, and ST78 versus KPC-Kp ST11, ST18, and ST258 strains analyzed here showed no statistically significant difference. These assays evidenced that high-risk clones of KPC-ECC and KPC-Kp can coexist in the same hospital environment including the same patient, which explains from an ecological point of view that both species can exchange and share plasmids. These findings offer hints to explain the worldwide rise of KPC-ECC strains based on the ability of some pandemic clones to compete and occupy a certain niche. Taken together, the presence of the same new plasmid and the fitness results that showed that both strains can coexist within the same patient suggest that horizontal genetic transfer of bla KPC-2 within the patient cannot be ruled out. These findings highlight the constant interaction that these two species can keep in the hospital environment, which, in turn, can be related to the spread of KPC.

This study aimed to identify the species of Enterobacter cloacae complex (ECC) isolates and compare the genotype, antibiotic resistance, and virulence among them. A total of 183 ECC isolates were collected from patients in eight hospitals in South Korea. Based on partial sequences of hsp60 and phylogenetic analysis, all ECC isolates were identified as nine species and six subspecies. Enterobacter hormaechei was the predominant species (47.0%), followed by Enterobacter kobei, Enterobacter asburiae, Enterobacter ludiwigii, and Enterobacter roggenkampii. Multilocus sequence typing analysis revealed that dissemination was not limited to a few clones, but E. hormaechei subsp. xiangfangensis, E. hormaechei subsp. steigerwaltii, and E. ludwigii formed large clonal complexes. Antibiotic resistance rates were different between the ECC species. In particular, E. asburiae, E. kobei, E. roggenkampii, and E. cloacae isolates were highly resistant to colistin, whereas most E. hormaechei and E. ludwigii isolates were susceptible to colistin. Virulence was evaluated through serum bactericidal assay and the Galleria mellonella larvae infection model. Consistency in the results between the serum resistance and the G. mellonella larvae infection assay was observed. Serum bactericidal assay showed that E. hormaechei, E. kobei, and E. ludwigii were significantly more virulent than E. asburiae and E. roggenkampii. In this study, we identified the predominant ECC species in South Korea and observed the differences in antibiotic resistance and virulence between the species. Our findings suggest that correct species identification, as well as continuous monitoring is crucial in clinical settings.

Due to the lack of research on the characteristics of different clusters of Enterobacter cloacae complex (ECC), this study aimed to characterize and explore the differences among species of the ECC. An analysis based on hsp60 showed that Enterobacter hormaechei was predominant in ECC. Interestingly, the antibiotic resistance rates of clusters were different, among which E. hormaechei subsp. steigerwaltii (cluster VIII) and Enterobacter cloacae IX (cluster IX) possessed high resistant rates to ciprofloxacin and levofloxacin, but cluster II (Enterobacter kobei) had low resistant rates. Cluster II exhibited a strong biofilm formation ability. Different motility and protease production ability were shown for distinct clusters. A PCR analysis showed that clusters I, III, VI, VIII, and IX carried more virulence genes, while cluster II had fewer. Clusters I, VIII, and IX with high pathogenicity were evaluated using the Galleria mellonella infection model. Thus, the characteristics of resistance, biofilm-forming ability, mobility, and virulence differed among the clusters. The strains were divided into 12 subgroups based on hsp60. The main clusters of ECC clinical strains were I, II, III, VI, VIII, and IX, among which IX, VIII, and I were predominant with high resistance and pathogenicity, and cluster II (E. kobei) was a special taxon with a strong biofilm formation ability under nutrient deficiency, but was associated with low resistance, virulence, and pathogenicity. Hence, clinical classification methods to identify ECC subgroups are an urgent requirement to guide the treatment of clinical infections.

To genetically characterize VIM-producing Enterobacter cloacae complex (ECC) isolates recovered in France from 2015 to 2018. WGS, species determination, MLST, clonal relationship and genetic characterization were performed on 149 VIM-producing ECC isolates. Among VIM-producing Enterobacterales, the prevalence of ECC increased drastically from 6% in 2012 to 52% in 2018. The most prevalent species were Enterobacter hormaechei subsp. hoffmannii (40.9%), E. hormaechei subsp. steigerwaltii (21.5%), E. hormaechei subsp. xiangfangensis (14.8%) and ECC clade S (17.4%). Major STs were ST-873 (17.5%), ST-66 (12.1%), ST-78 (9.4%), ST-419 (8.1%), ST-145 (4.7%), ST-50 (4.0%), ST-118 (4.0%) and ST-168 (4.0%). Finally, six different integrons were identified, with some being specific to a given blaVIM variant (In916 with blaVIM-1-aacA4'-aphA15-aadA1-catB2 and In416 with blaVIM-4-aacA7-dfrA1b-aadA1b-smr2 genes). This study demonstrated the genetic diversity among VIM-producing ECC isolates, indicating that their spread is not linked to a single clone.

Biofilms pose a significant threat to public health due to their role in antibiotic-resistant infections, including urinary tract infections, cystic fibrosis, and infective endocarditis. Enterobacter hormaechei, a Gram-negative bacterium within the Enterobacter cloacae complex (ECC), is a known biofilm-former that contributes to infections in the urinary tract, soft tissues, and medical devices. This study investigates the antibiofilm activity of naringin (NA), a flavonoid derived from naringenin, against E. hormaechei using various assays. Minimum Inhibitory Concentration (MIC) assay revealed that NA inhibits planktonic bacterial growth, with an MIC value of 4.096 mg/mL. Crystal Violet (CV) assay revealed a significant reduction in biofilm formation at NA concentrations of 0.5 mg/mL, 1 mg/mL, and 1.5 mg/mL compared to controls. Fluorescence microscopy further confirmed a decrease in bacterial population and disruption of biofilm architecture following NA treatment. In silico analysis was conducted to investigate the potential molecular interactions of NA with the biofilm regulatory proteins MrkB and FimA. The results indicated that NA might effectively inhibit biofilm formation in E. hormaechei by targeting these two key proteins involved in pilus biogenesis and bacterial adherence. These findings suggest that NA could serve as a potential therapeutic agent against E. hormaechei-associated infections.

The emergence of multidrug-resistant (MDR) bacterial strains is one of the significant global challenges with regard to bacterial drug-resistance control. Enterobacter hormaechei organisms belong to the Enterobacter cloacae complex (ECC) and are commonly recognized as causative agents for hospital infections. Recently, a few E. hormaechei MDR strains associated with infection in piglets, calves, and a fox were reported, highlighting the important role of animals and livestock in the emergence and spread of antimicrobial resistance. In this study, the vaginal swab sample from a 5-year-old cow with multiple anamnestic infectious abortions was carefully investigated. The animal was unresponsive to antibiotic therapy recommended by the veterinarian. The MDR bacterial strain isolated from the bovine sample, designated as the Saratov_2019, belonged to Enterobacter hormaechei. The genome-based phylogenetic analysis identified the isolate to be Enterobacter hormaechei subsp. xiangfangensis. The genome of the Saratov_2019 contained a 6364 bp plasmid. Importantly, we revealed the novel sequence type ST1416 and 13 MDR genes correlating with the MDR phenotype in only the chromosome but not the plasmid. These findings indicate that the potential spread of this strain may pose a threat for both animal and human health. The data obtained here support the notion of the important role of livestock in the emergence and spread of antimicrobial resistance, promoting careful investigation of the MDR spectra for livestock-related bacterial isolates. To the best of our knowledge, this is the first report on the association of E. hormaechei subsp. xiangfangensis with the infection of the reproductive system in cattle.