Multiple resistance factors collectively promote inoculum-dependent dynamic survival during antimicrobial peptide exposure in Enterobacter cloacae.

Antimicrobial peptides (AMPs) are a promising tool with which to fight rising antibiotic resistance. However, pathogenic bacteria are equipped with several AMP defense mechanisms, whose contributions to AMP resistance are often poorly defined. Here, we evaluate the genetic determinants of resistance to an insect AMP, cecropin B, in the opportunistic pathogen Enterobacter cloacae. Single-cell analysis of E. cloacae's response to cecropin revealed marked heterogeneity in cell survival, phenotypically reminiscent of heteroresistance (the ability of a subpopulation to grow in the presence of supra-MIC concentration of antimicrobial). The magnitude of this response was highly dependent on initial E. cloacae inoculum. We identified 3 genetic factors which collectively contribute to E. cloacae resistance in response to the AMP cecropin: The PhoPQ-two-component system, OmpT-mediated proteolytic cleavage of cecropin, and Rcs-mediated membrane stress response. Altogether, our data suggest that multiple, independent mechanisms contribute to AMP resistance in E. cloacae.

IMI-Type Carbapenemase-Producing Enterobacter cloacae Complex, France and Overseas Regions, 2012-2022.

We characterized a collection of IMI-like-producing Enterobacter spp. isolates (n = 112) in France. The main clone corresponded to IMI-1-producing sequence type 820 E. cloacae subspecies cloacae that was involved in an outbreak. Clinicians should be aware of potential antimicrobial resistance among these bacteria.

Clinical and genomic characteristics of IMP-producing Enterobacter cloacae complex and Klebsiella pneumoniae.

Carbapenemase-producing Enterobacterales (CPEs) are one of the top priority antimicrobial-resistant pathogens. Among CPEs, those producing acquired metallo-ß-lactamases (MBLs) are considered particularly problematic as few agents are active against them. Imipenemase (IMP) is the most frequently encountered acquired MBL in Japan, but comprehensive assessment of clinical and microbiological features of IMP-producing Enterobacterales infection remains scarce. Here, we retrospectively evaluated 62 patients who were hospitalized at a university hospital in Japan and had IMP-producing Enterobacterales from a clinical culture. The isolates were either Enterobacter cloacae complex or Klebsiella pneumoniae, and most of them were isolated from sputum. The majority of K. pneumoniae, but not E. cloacae complex isolates, were susceptible to aztreonam. Sequence type (ST) 78 and ST517 were prevalent for E. cloacae complex and K. pneumoniae, respectively, and all isolates carried blaIMP-1. Twenty-four of the patients were deemed infected with IMP-producing Enterobacterales. Among the infected patients, therapy varied and largely consisted of conventional ß-lactam agents, fluoroquinolones, or combinations. Three (13%), five (21%), and nine (38%) of them died by days 14, 30, and 90, respectively. While incremental mortality over 90 days was observed in association with underlying comorbidities, active conventional treatment options were available for most patients with IMP-producing Enterobacterales infections, distinguishing them from more multidrug-resistant CPE infections associated with globally common MBLs, such as New Delhi metallo-ß-lactamase (NDM) and Verona integron-encoded metallo-ß-lactamase (VIM).

Hybrid-genome sequence analysis of Enterobacter cloacae FACU and morphological characterization: insights into a highly arsenic-resistant strain.

Many organisms have adapted to survive in environments with high levels of arsenic (As), a naturally occurring metalloid with various oxidation states and a common element in human activities. These organisms employ diverse mechanisms to resist the harmful effects of arsenic compounds. Ten arsenic-resistant bacteria were isolated from contaminated wastewater in this study. The most efficient bacterial isolate able to resist 15,000 ppm Na2HAsO4·7H2O was identified using the 16S rRNA gene and whole genome analysis as Enterobacter cloacae FACU. The arsenic E. cloacae FACU biosorption capability was analyzed. To further unravel the genetic determinants of As stress resistance, the whole genome sequence of E. cloacae FACU was performed. The FACU complete genome sequence consists of one chromosome (5.7 Mb) and two plasmids, pENCL 1 and pENCL 2 (755,058 and 1155666 bp, respectively). 7152 CDSs were identified in the E. cloacae FACU genome. The genome consists of 130 genes for tRNA and 21 for rRNAs. The average G + C content was found to be 54%. Sequencing analysis annotated 58 genes related to resistance to many heavy metals, including 16 genes involved in arsenic efflux transporter and arsenic reduction (five arsRDABC genes) and 42 genes related to lead, zinc, mercury, nickel, silver, copper, cadmium and chromium in FACU. Scanning electron microscopy (SEM) confirmed the difference between the morphological responses of the As-treated FACU compared to the control strain. The study highlights the genes involved in the mechanism of As stress resistance, metabolic pathways, and potential activity of E. cloacae FACU at the genetic level.

High-level carbapenem tolerance requires antibiotic-induced outer membrane modifications.

Antibiotic tolerance is an understudied potential contributor to antibiotic treatment failure and the emergence of multidrug-resistant bacteria. The molecular mechanisms governing tolerance remain poorly understood. A prominent type of ß-lactam tolerance relies on the formation of cell wall-deficient spheroplasts, which maintain structural integrity via their outer membrane (OM), an asymmetric lipid bilayer consisting of phospholipids on the inner leaflet and a lipid-linked polysaccharide (lipopolysaccharide, LPS) enriched in the outer monolayer on the cell surface. How a membrane structure like LPS, with its reliance on mere electrostatic interactions to maintain stability, is capable of countering internal turgor pressure is unknown. Here, we have uncovered a novel role for the PhoPQ two-component system in tolerance to the ß-lactam antibiotic meropenem in Enterobacterales. We found that PhoPQ is induced by meropenem treatment and promotes an increase in 4-amino-4-deoxy-L-aminoarabinose [L-Ara4N] modification of lipid A, the membrane anchor of LPS. L-Ara4N modifications likely enhance structural integrity, and consequently tolerance to meropenem, in several Enterobacterales species. Importantly, mutational inactivation of the negative PhoPQ regulator mgrB (commonly selected for during clinical therapy with the last-resort antibiotic colistin, an antimicrobial peptide [AMP]) results in dramatically enhanced tolerance, suggesting that AMPs can collaterally select for meropenem tolerance via stable overactivation of PhoPQ. Lastly, we identify histidine kinase inhibitors (including an FDA-approved drug) that inhibit PhoPQ-dependent LPS modifications and consequently potentiate meropenem to enhance lysis of tolerant cells. In summary, our results suggest that PhoPQ-mediated LPS modifications play a significant role in stabilizing the OM, promoting survival when the primary integrity maintenance structure, the cell wall, is removed.

In vitro and in vivo activity of ceftazidime/avibactam and aztreonam alone or in combination against mcr-9, serine- and metallo-ß-lactamases-co-producing carbapenem-resistant Enterobacter cloacae complex.

PURPOSE: Enterobacteriaceae carrying mcr-9, in particularly those also co-containing metallo-ß-lactamase (MBL) and TEM type ß-lactamase, present potential transmission risks and lack adequate clinical response methods, thereby posing a major threat to global public health. The aim of this study was to assess the antimicrobial efficacy of a combined ceftazidime/avibactam (CZA) and aztreonam (ATM) regimen against carbapenem-resistant Enterobacter cloacae complex (CRECC) co-producing mcr-9, MBL and TEM. METHODS: The in vitro antibacterial activity of CZA plus ATM was evaluated using a time-kill curve assay. Furthermore, the in vivo interaction between CZA plus ATM was confirmed using a Galleria mellonella (G. mellonella) infection model. RESULTS: All eight clinical strains of CRECC, co-carrying mcr-9, MBL and TEM, exhibited high resistance to CZA and ATM. In vitro time-kill curve analysis demonstrated that the combination therapy of CZA + ATM exerted significant bactericidal activity against mcr-9, MBL and TEM-co-producing Enterobacter cloacae complex (ECC) isolates with a 100% synergy rate observed in our study. Furthermore, in vivo survival assay using Galleria mellonella larvae infected with CRECC strains co-harboring mcr-9, MBL and TEM revealed that the CZA + ATM combination significantly improved the survival rate compared to the drug-treatment alone and untreated control groups. CONCLUSION: To our knowledge, this study represents the first report on the in vitro and in vivo antibacterial activity of CZA plus ATM against CRECC isolates co-harboring mcr-9, MBL and TEM. Our findings suggest that the combination regimen of CZA + ATM provides a valuable reference for clinicians to address the increasingly complex antibiotic resistance situation observed in clinical microorganisms.

Diversity of MrTolls and their regulation of antimicrobial peptides expression during Enterobacter cloacae infection in Macrobrachium rosenbergii.

Toll/Toll-like receptor (TLR) is an important pattern recognition receptor that plays an important role in the immunity of animals. Six Toll genes were identified in Macrobrachium rosenbergii, namely, MrToll, MrToll1, MrToll2, MrToll3, MrToll4, and MrToll5. SMART analysis showed that all six Tolls have a transmembrane domain, a TIR domain, and different number of LRR domains. The phylogenetic tree showed that six Tolls were located in six different branches. Among these six Tolls, only MrToll4 contains the QHR motif, which is similar to insect Toll9. MrToll4 belongs to V-type/scc Toll with only one LRRCT domain. MrToll1 and MrToll5 are classical P-type/mcc Toll with two LRRCT domains and an LRRNT. MrTolls were distributed in the hemocytes, heart, hepatopancreas, gills, stomach, and intestine. During the infection of Enterobacter cloacae, the expression level of MrToll and MrToll1-4 was upregulated in the intestine of M. rosenbergii. RNA interference experiments showed that the expression of most antimicrobial peptide (AMP) genes was negatively regulated by MrTolls during E. cloacae infection. On the contrary, crustin (Cru) 3 and Cru4 were inhibited after the knockdown of MrToll, and Cru1 and Cru4 were significantly downregulated with the knockdown of MrToll4 during E. cloacae challenge. These results suggest that MrTolls may be involved in the regulation of AMP expression in the intestine during E. cloacae infection.

Some virulence genes are associated with antibiotic susceptibility in Enterobacter cloacae complex.

BACKGROUND: Enterobacter cloacae complex (ECC) including different species are isolated from different human clinical samples. ECC is armed by many different virulence genes (VGs) and they were also classified among ESKAPE group by WHO recently. The present study was designed to find probable association between VGs and antibiotic susceptibility in different ECC species. METHODS: Forty-five Enterobacter isolates that were harvested from different clinical samples were classified in four different species. Seven VGs were screened by PCR technique and antibiotic susceptibility assessment was performed by disk-diffusion assay. RESULT: Four Enterobacter species; Enterobacter cloacae (33.3%), Enterobacter hormaechei (55.6%), Enterobacter kobei (6.7%) and Enterobacter roggenkampii (4.4%) were detected. Minimum antibiotic resistance was against carbapenem agents and amikacin even in MDR isolates. 33.3% and 13.3% of isolates were MDR and XDR respectively. The rpoS (97.8%) and csgD (11.1%) showed maximum and minimum frequency respectively. Blood sample isolated were highly virulent but less resistant in comparison to the other sample isolates. The csgA, csgD and iutA genes were associated with cefepime sensitivity. CONCLUSION: The fepA showed a predictory role for differentiating of E. hormaechei from other species. More evolved iron acquisition system in E. hormaechei was hypothesized. The fepA gene introduced as a suitable target for designing novel anti-virulence/antibiotic agents against E. hormaechei. Complementary studies on other VGs and ARGs and with bigger study population is recommended.

First Record of the Isolation of blaNDM-1-Producing Enterotoxigenic Enterobacter cloacae ssp. cloacae in Baghdad/Iraq.

BACKGROUND: Antibiotic resistance is a major problem threatening human beings. The genetic determinants that carry resistance genes can be transmitted in several ways in clinical and food environments. Hence, this research study aimed to investigate the presence of New Delhi metallo-beta-lactamase-1 (blaNDM-1) produced by enterotoxigenic Enterobacter cloacae in both clinical and food samples. METHODS AND RESULTS: Twenty-four isolates of Enterobacter spp. were isolated, seven isolates from food samples and 17 isolates from blood taken from neonates and children (1 day - 10 years old) resident in a children's hospital. Antibiotic susceptibility test to 14 antibiotics was performed for all isolates. Enterotoxigenicity of the clinical and foodborne isolates was detected phenotypically using Suckling mouse bioassay. Genomic deoxyribonucleic acid (DNA) was extracted from the isolated Enterobacter spp. that were detected resistant to imipenem. Polymerase chain reaction (PCR) was used to amplify blaNDM-1 gene followed by sequencing. The results of the bioassay revealed that 64.28% of E. cloacae ssp. cloacae isolates were enterotoxigenic. Two E. cloacae ssp. cloacae were imipenem resistant. CONCLUSIONS: This study showed that one isolate from a male child 1 < year was bla NDM-1 positive that was con-firmed by sequencing. This is the first report that revealed blaNDM-1 producing Enterobacter cloacae in Iraq.

Molecular Characterization of Class 1 Integrons and Carbapenem-Resistant Genes in Enterobacter cloacae Complex Isolates.

Enterobacter cloacae complex (ECC) widely exists in the hospital environment and is one of the important conditional pathogens of hospital-acquired infection. To investigate the distribution of integrons and carbapenem-resistant genes in clinical ECC, 70 isolates of ECC from non-sputum specimens were collected. Class 1 and class 2 integron integrase gene intI1 and intI2, as well as common carbapenem-resistant genes, blaKPC, blaVIM, blaIMP, blaNDM, blaGES, and blaOXA-23, were screened. Gene cassette arrays and common promoters of class 1 integron together with subtypes of carbapenem-resistant genes were determined by sequencing. Resistant rates to commonly used antimicrobial agents between class 1 integron-positive and integron-negative ECC isolates were analyzed. The whole-genome of blaNDM-7 harboring Enterobacter hormaechei was sequenced and the sequence around blaNDM-7 was analyzed. Twenty isolates were positive for intI1. Nineteen different antimicrobial-resistant gene cassettes and 11 different gene cassette arrays, including aadA22-lnuF, were detected in this study. Common promoters of class 1 integron PcH1, PcW, PcW-P2, and PcH2 were detected in 12, 4, 3, and 1 isolates, respectively. The rates of antimicrobial resistance of intI1-positive isolates were higher than those of intI1-negative isolates to clinical commonly used antimicrobial agents. Carbapenem-resistant genes blaKPC-2, blaNDM-1, blaNDM-2, and blaNDM-7 were detected in 2, 1, 1, and 1 isolates, respectively. blaNDM-7 was located between bleMBL and IS5. To the best of our knowledge, this study reported for the first time of blaNDM-7 in ECC isolate in China.

[Analysis of gene characteristics and core genome characteristics of carbapenem-resistant Enterobacter cloacae in rural residents of Weifang City, Shandong Province].

Objective: To investigate the drug-resistant gene characteristics and core genome characteristics of carbapenem-resistant Enterobacter cloacae (CR-ECL) in rural residents of Weifang City, Shandong Province. Methods: Fecal samples were collected from rural community residents in Weifang City, Shandong Province in 2017. Drug-resistant strains were screened using a carbapenem-resistant enterobacter chromogenic medium. CR-ECL positive strains were acquired via Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry(MALDI-TOFMS) analysis. The antibiotic resistance phenotype of CR-ECL was determined using a microbroth dilution assay. Whole genome sequencing (WGS) and analysis were conducted, along with an examination of the immediate vicinity of the blaNDM gene and phylogenetic analysis of the strains. Results: A total of 628 fecal samples were collected and tested, of which 6 were CR-ECL positive (detection rate 0.96%), all exhibiting multiple drug resistance (MDR) phenotypes. Six CR-ECL strains had four MLST genotypes (ST), all of which carried multiple drug resistance genes (blaNDM-1, blaNDM-5, etc.) and virulence genes (acrA, acrB, entB, fepC, etc.). There were mobile genetic elements ISAba125, TN3-IS3000, TN3 and IS5 in the genetic environment surrounding the blaNDM gene. The phylogenetic tree showed that the multi-locus sequence typing of the core genome (cgMLST) was consistent with the single nucleotide polymorphism (SNPs) results. The cgMLST results showed that the allele differences between strains 2BC0101B and 2BC0251B, 2BG0561B and 2BI0221B were 2 and 1, respectively. The SNPs results showed that the above two pairs of bacteria also clustered together. It was found that the strains of chicken fecal samples in the National Center for Biotechnology Information (NCBI) database were located in the center of the evolutionary tree, and the local sequences could be traced back to American human sequences. Conclusion: Multidrug-resistant CR-ECL is detected in rural community residents in Weifang City, Shandong Province.

Temocillin Resistance in the Enterobacter cloacae Complex Is Conferred by a Single Point Mutation in BaeS, Leading to Overexpression of the AcrD Efflux Pump.

The Enterobacter cloacae complex (ECC) has become a major opportunistic pathogen with antimicrobial resistance issues. Temocillin, an "old" carboxypenicillin that is remarkably stable toward ß-lactamases, has been used as an alternative for the treatment of multidrug-resistant ECC infections. Here, we aimed at deciphering the never-investigated mechanisms of temocillin resistance acquisition in Enterobacterales. By comparative genomic analysis of two clonally related ECC clinical isolates, one susceptible (Temo_S [MIC of 4 mg/L]) and the other resistant (Temo_R [MIC of 32 mg/L]), we found that they differed by only 14 single-nucleotide polymorphisms, including one nonsynonymous mutation (Thr175Pro) in the two-component system (TCS) sensor histidine kinase BaeS. By site-directed mutagenesis in Escherichia coli CFT073, we demonstrated that this unique change in BaeS was responsible for a significant (16-fold) increase in temocillin MIC. Since the BaeSR TCS regulates the expression of two resistance-nodulation-cell division (RND)-type efflux pumps (namely, AcrD and MdtABCD) in E. coli and Salmonella, we demonstrated by quantitative reverse transcription-PCR that mdtB, baeS, and acrD genes were significantly overexpressed (15-, 11-, and 3-fold, respectively) in Temo_R. To confirm the role of each efflux pump in this mechanism, multicopy plasmids harboring mdtABCD or acrD were introduced into either Temo_S or the reference strain E. cloacae subsp. cloacae ATCC 13047. Interestingly, only the overexpression of acrD conferred a significant increase (from 8- to 16-fold) of the temocillin MIC. Altogether, we have shown that temocillin resistance in the ECC can result from a single BaeS alteration, likely resulting in the permanent phosphorylation of BaeR and leading to AcrD overexpression and temocillin resistance through enhanced active efflux.

X-ray structures of Enterobacter cloacae allose-binding protein in complexes with monosaccharides demonstrate its unique recognition mechanism for high affinity to allose.

d-Allose is an aldohexose of the C3-epimer of d-glucose, existing in very small amounts in nature, called a rare sugar. The operon responsible for d-allose metabolism, the allose operon, was found in several bacteria, which consists of seven genes: alsR, alsB, alsA, alsC, alsE, alsK, and rpiB. To understand the biological implication of the allose operon utilizing a rare sugar of d-allose as a carbon source, it is important to clarify whether the allose operon functions specifically for d-allose or also functions for other ligands. It was proposed that the allose operon can function for d-ribose, which is essential as a component of nucleotides and abundant in nature. Allose-binding protein, AlsB, coded in the allose operon, is thought to capture a ligand outside the cell, and is expected to show high affinity for the specific ligand. X-ray structure determinations of Enterobacter cloacae AlsB (EtcAlsB) in ligand-free form, and in complexes with d-allose, d-ribose, and d-allulose, and measurements of the thermal parameters of the complex formation using an isothermal titration calorimeter were performed. The results demonstrated that EtcAlsB has a unique recognition mechanism for high affinity to d-allose by changing its conformation from an open to a closed form depending on d-allose-binding, and that the binding of d-ribose to EtcAlsB could not induce a completely closed form but an intermediate form, explaining the low affinity for d-ribose.

Antimicrobial-Resistant Enterobacterales Recovered from the Environment of Two Zoological Institutions Include Enterobacter cloacae Complex ST171 Producing KPC-4 Carbapenemase.

Environmental surfaces can serve as reservoirs for pathogens and antimicrobial-resistant (AMR) bacteria in healthcare settings. Although active surveillance programs are used in veterinary and human healthcare, unconventional settings like zoological facilities are often overlooked, even though antimicrobials are used to maintain the health of their animal collections. Here, we used electrostatic cloths to conduct active environmental surveillance over a 2-year period at two zoological institutions to determine contamination prevalence of human-only and mixed animal-human touch environments with AMR bacteria. We recovered Enterobacterales isolates that expressed quinolone resistance, an AmpC-like phenotype, and an extended-spectrum ß-lactamase phenotype from 144 (39%), 141 (38.2%), and 72 (19.5%) of the environmental samples, respectively. The zoological institutions, areas and exhibits within the zoological facility, and sampling surface type affected the odds of recovering AMR bacteria from the environment. Three carbapenemase-producing Enterobacter cloacae complex ST171 isolates recovered from one zoological facility harbored an IncH12 plasmid with a Tn4401b-KPC-4 transposon conferring multidrug resistance. One isolate maintained three tandem repeats of a Tn4401b-KPC-4 element on an IncHI2 plasmid, although this isolate was susceptible to the four carbapenem drugs tested. These three isolates and their IncH12 plasmids shared significant genomic similarity with two E. cloacae complex isolates recovered from canine patients at a regional veterinary hospital during year 2 of this study. Our results indicated that surface environments at zoological institutions can serve as reservoirs for AMR bacteria and their genes and have implications for animal and public health. IMPORTANCE Environmental surfaces can be a source of antimicrobial-resistant (AMR) bacteria that pose a risk to human and animal health. Zoological institutions are unique environments where exotic animals, staff, and visitors intermingle and antimicrobials are used to maintain animal health. However, zoological environments are often overlooked as reservoirs of AMR bacteria. Here, we show that zoological environments can serve as reservoirs of fluoroquinolone-resistant and extended-spectrum cephalosporin-resistant bacteria. In addition, we isolated three carbapenemase-producing Enterobacter cloacae complex strains carrying blaKPC-4, including one with a unique, tandem triplicate of the Tn4401b-KPC-4 element. Comparative whole genomics of these isolates with two E. cloacae complex isolates from patients at a regional veterinary hospital highlighted the possibility of local KPC-4 spread between animal environments. Our results suggest that environments at zoological institutions serve as reservoirs for AMR bacteria and pose a hypothetical One Health risk to the public, staff, and the wild animal populations in captivity.

Molecular eidemiology of carbapenem-resistant Enterobacter cloacae complex in a tertiary hospital in Shandong, China.

BACKGROUND: The increasing incidence and prevalence of carbapenem-resistant Enterobacter cloacae complex (CREC) poses great challenges to infection prevention and disease treatment. However, much remains unknown about the clinical characteristics of CREC isolates. Our objective was to characterize antimicrobial resistance and, carbapenemase production in CREC with 36 CREC isolates collected from a tertiary hospital in Shandong, China. RESULTS: Three types of carbapenemases (NDM, IMP and VIM) were detected in these isolates. Among them, NDM carbapenemases were most prevalent, with a 61.2% (22/36) detection rate for NDM-1, 27.8% (10/36) for NDM-5 and 2.8% (1/36) for NDM-7. IMP-4 was found in two isolates and VIM-1 in only one isolate. The MLST analysis identified 12 different sequence types (STs), of which ST171 (27.8%) was the most prevalent, followed by ST418 (25.0%). ST171 isolates had significantly higher rates of resistance than other STs to gentamicin and tobramycin (Ps < 0.05), and lower rates of resistance to aztreonam than ST418 and other STs (Ps < 0.05). Among 17 carbapenemase-encoding genes, the blaNDM-5 gene was more frequently detected in ST171 than in ST418 and other isolates (Ps < 0.05). In contrast, the blaNDM-1 gene was more frequently seen in ST418 than in ST171 isolates. One novel ST (ST1965) was identified, which carried the blaNDM-1 gene. CONCLUSION: NDM-5 produced by ST171 and NDM-1 carbapenemase produced by ST418 were the leading cause of CREC in this hospital. This study enhances the understanding of CREC strains and helps improve infection control and treatment in hospitals.

Transcriptomic profiling and characterization of microRNAs in Macrobrachium rosenbergii potentially involved in immune response to Enterobacter cloacae infection.

Enterobacter cloacae is a member of the Enterobacter family, which could prevent Macrobrachium rosenbergii from growing and cause mass mortality. However, no research has focused on microRNA immunity in M. rosenbergii infected with E. cloacae. To clarify the immune response mechanisms, transcriptomic analysis was performed on the miRNAs of M. rosenbergii infected with E. cloacae YZ3 strain. Following quality screening, 10,616,712 clean reads were obtained from the control group and 12,726,421 from the infected group. Among 899 known miRNAs, 446 differentially expressed miRNAs (DEMs) were identified. Meanwhile, 59 novel miRNAs were predicted, along with 39 DEMs. Target genes of DEMs have been predicted in order to gain a deeper understanding of the immune-related functions. GO and KEGG pathway analysis revealed the biological functions and signaling pathways of target genes. The results indicated that E. cloacae significantly affected the NOD-like receptor, RIG-I-like receptor and Toll-like receptor pathways. Ten DEMs were randomly selected, and their expression level was verified by Quantitative Real-time PCR technology. Overall, this study highlights the influential role of miRNAs in the innate immune system of M. rosenbergii, which has important implications for developing new strategies to prevent and treat related diseases in the future.

Comparative genome analyses uncovered the cadmium resistance mechanism of enterobacter cloacae.

Cadmium (Cd) can be transported into plants from polluted soils and may cause animal and human diseases through food chains, which requires the development of highly efficient methods for soil Cd remediation. Although we isolated an Enterobacter cloacae strain Cu6 with Cd resistance, this strain cannot be used for soil Cd remediation due to its lower resistance. Here, we domesticated Cu6 and obtained a highly Cd-resistant strain, LPY6, and found that this strain can attenuate the toxic effects of Cd on wheat seedling growth. We deciphered the high Cd-resistance mechanism of LPY6 by genome comparative and genetic analysis. Compared with Cu6, 75 genes were mutated in LPY6. Thirty-four of these genes were deleted, and 41 had single nucleotide polymorphisms (SNPs). Most of these mutated proteins are involved in basic metabolism, substrate transport, stress response and formate and hydrogen metabolism. RNA quantitative analysis and promoter activity assays showed that the transcription or mRNA levels of two operons (cadA and norVW) in these mutated genes were regulated by Cd, zinc (Zn) or lead (Pb) ions, suggesting that these two operons might be required for Cd, Zn or Pb resistance. Expression of cadA and norVW operons in LPY6 partially recovered Cd susceptibility, demonstrating that CadA and NorVW are involved in Cd resistance in E. cloacae. Our findings illustrate that E. cloacae acquires Cd resistance through different pathways and lay a foundation for developing highly efficient methods for soil Cd remediation.

Outbreak of OXA-48-producing Enterobacteriaceae in a neonatal intensive care unit in Western Sweden.

In 2015, an outbreak caused by OXA-48-producing Enterobacteriaceae affected a neonatal intensive care unit at a Swedish University Hospital. The aim was to explore the transmission of OXA-48-producing strains between infants and the transfer of resistance plasmids between strains during the outbreak. Twenty-four outbreak isolates from ten suspected cases were whole-genome sequenced. A complete assembly was created for the index isolate (Enterobacter cloacae) and used as a mapping reference to detect its plasmids in the remaining isolates (17 Klebsiella pneumoniae, 4 Klebsiella aerogenes, and 2 Escherichia coli). Strain typing was performed using core genome MLST and SNP analysis. As judged from sequencing and clinical epidemiological data, the outbreak involved nine cases (two developed sepsis) and four OXA-48-producing strains: E. cloacae ST1584 (index case), K. pneumoniae ST25 (eight cases), K. aerogenes ST93 (two cases), and E. coli ST453 (2 cases). Two plasmids from the index strain, pEclA2 and pEclA4, carrying blaOXA48 and blaCMY-4, respectively, were traced to all K. pneumoniae ST25 isolates. Klebsiella aerogenes ST93 and E. coli ST453 harboured either only pEclA2, or both pEclA2 and pEclA4. One suspected case harbouring OXA-162-producing K. pneumoniae ST37 could be excluded from the outbreak. Once initiated by an E. cloacae strain, the outbreak was caused by the dissemination of a K. pneumoniae ST25 strain and involved inter-species horizontal transfer of two resistance plasmids, one of which carried blaOXA-48. To our knowledge, this is the first description of an outbreak of OXA-48-producing Enterobacteriaceae in a neonatal setting in northern Europe.

The occurrence and genomic characteristics of the blaIMI-1 carbapenemase-producing Enterobacter cloacae complex retrieved from natural water sources in central Thailand.

AIM: Carbapenem resistance among Enterobacteriaceae is a serious threat to humans worldwide. This study aims to evaluate the phenotypic and genotypic characterization of carbapenemase-producing Enterobacter cloacae complex (ECC) retrieved from water sources in the central part of Thailand. METHODS AND RESULTS: Samples were collected from water bodies surrounding farms and communities in central Thailand. The species were identified by using MALDI-TOF MS. The minimum inhibitory concentration (MIC) and antibiotic susceptibility were determined. The carbapenemase-producing genes were detected by PCR and whole genome sequencing (WGS). ECC with chromosome-encoded blaIMI-1 carbapenemase were detected. These isolates were resistant to last-resort antibiotics such as carbapenems and colistin as well as penicillin. In addition, all blaIMI-1 genes isolated from this study were found to be associated with chromosomally integrated Xer-dependent integrative mobile elements (IMEXs). CONCLUSION: These findings highlight the diversity and dissemination of carbapenemases-producing Enterobacterales in environmental sources. With the increasing detection of carbapenemase genes worldwide, we should be aware of the blaIMI-producing E. cloacae complex with a high resistance profile and the ability to mobilize within the environment.

Detection of clinically relevant carbapenemase encoding genes in carbapenem-resistant Enterobacter cloacae complex and Klebsiella pneumoniae isolated from farmed freshwater fish.

AIMS: The present study was aimed to detect clinically relevant carbapenemase encoding genes in carbapenem-resistant Enterobacter cloacae complex (CR-ECC), Klebsiella pneumoniae (CR-KP), and Serratia plymuthica (CR-SP) isolated from farmed freshwater fish. METHODS AND RESULTS: Out of 243 spatially diverse freshwater fish samples analysed, 5.3% were contaminated with CR-ECC, 1.6% with CR-KP, and 0.4% with CR-SP. The CR-ECC was further identified as E. asburiae (38.5%), E. mori (23.1%), E. cloacae (15.4%), E. hormaechei (15.4%), and E. kobei (7.7%) by 16S rRNA gene sequencing. The CR-ECC were resistant to carbapenems and cefoxitin, whereas CR-KP and CR-SP were multi-drug resistant (MDR). The CR-ECC harboured the carbapenemase gene blaIMI alone or in combination with blaTEM, blaEBC, blaCIT, blaACC, and tet(E). Whereas, CR-KP harboured carbapenemase gene, blaNDM-5 along with blaOXA-48, blaSHV, blaOXA-1, blaCTX-M-15, tet(A), sul1, and qnrB. No carbapenemase-encoding genes were detected in CR-SP. The MLST analysis showed that CR-KP belonged to ST231 and ST1561 lineages, while CR-ECC did not show exact match with any reported STs. The plasmid replicons predominantly detected were IncF and IncI1. Broth mating assays of CR-KP and CR-ECC with recipient Escherichia coli J53 indicated that blaNDM-5 was transferable but not blaIMI. CONCLUSION: This study highlights the low-level contamination of carbapenem-resistant Enterobacterales (CRE) harbouring clinically relevant carbapenemase-encoding genes in farmed freshwater fish from India. The CR-ECC of fish origin did not show the potential to spread carbapenem resistance.