lsolation of Leclercia adecarboxylata Producing Carbapenemases in A Newborn Female＊

MENG Shuang ,MIAO Bei Bei ,LI Jie ,YIN Jian Wen ,LIU Ze Liang ,JIANG Xue Qi ,GONG Xin Yi,and LI Juan,#

Leclercia adecarboxylata is a Gram-negative bacterium belonging to the Enterobacteriaceae family.To our knowledge,this is the first report of a carbapenem-resistant L.adecarboxylata strain isolated from a healthy newborn.The L.adecarboxylata strain isolated in this study carried four plasmids that may serve as reservoirs for antibiotic resistance genes.Plasmids 2 and 4 did not harbor any antimicrobial resistance genes.Plasmid 3 is a novel plasmid containing three resistance genes.The blaIMP gene harbored in the strain was most similar to blaIMP-79 at the nucleotide level,with a similarity of 99.4% (737/741).This case highlights the importance of considering L.adecarboxylata as a potential cause of infections in children.

Key words: Leclercia adecarboxylata;Carbapenem-resistant;Newborn

Leclercia adecarboxylatais a motile,facultatively anaerobic,Gram-negative bacterium first identified by Leclerc in 1962[1].L.adecarboxylatahas been isolated from water,food,and other environmental sources,and is now recognized as a pathogenic organism[2].Previous studies have reported the presence of this bacterium in immunocompromised patients;however,several recent cases ofL.adecarboxylatahave been reported in immunocompetent patients,particularly children[3-5].

Carbapenems are first-line antibiotics used to treat multidrug-resistant Gram-negative bacterial infections.However,carbapenem-resistant Enterobacteriaceae have become a major public health threat,leading to severe infections,limited treatment options,and mortality rates of 26%–44%.The prevalence of carbapenem resistance in Enterobacteriaceae is mediated by the rapid emergence of carbapenemase genes[6,7].Although most cases ofL.adecarboxylatainfection are susceptible to common antibiotics,some drugresistant strains have recently been detected[8-10].A review of cases ofL.adecarboxylatainfection in humans revealed 82 publications describing clinical cases ofL.adecarboxylatainfection in 148 patients(104 adults and 44 children) since the first report in 1991[11-14].Among the documented cases of pediatric infection,one case occurred in 1991,two in 2000–2004,six in 2011–2014,four in 2015–2019,and 31 in 2020–2022.L.adecarboxylatainfection remains relatively rare;however,the number of reported cases in children has recently increased[15].Here,we present a case of a carbapenem-resistantL.adecarboxylatastrain in a newborn female to increase awareness ofL.adecarboxylataas an emerging infection in children.

L.adecarboxylata17YN198 was isolated in 2017 from the feces of a healthy 5-day-old female newborn with no relevant medical history in Yunnan,China.The strain was preliminarily identified using matrix-assisted laser desorption ionization time-offlight mass spectrometry (MALDI-TOF MS) (Bruker Daltonics,Billerica,MA,USA)[16].Identification was confirmed by 16S rRNA gene sequencing.Antimicrobial susceptibility was determined using the broth microdilution method,according to the Clinical Laboratory Standards Institute (CLSI)guidelines[17].Twenty-nine common antimicrobial agents were used to evaluate antimicrobial susceptibility. The isolated strain exhibited resistance to ceftriaxone, ceftazidime, cefazolin, amoxicillin-clavulanate,trimethoprimsulfamethoxazole,ertapenem, and meropenem(Table 1) according to the CLSI guidelines,and was susceptible to amikacin,gentamicin,aztreonam,chloramphenicol,norfloxacin,piperacillintazobactam,and minocycline.

Table 1.The MIC profile of 29 common antimicrobial agents for Leclercia adecarboxylata strain 17YN198

Whole-genome sequencing was performed using the Illumina NovaSeq PE150 platform (Illumina,https://www.illumina.com) and PacBio singlemolecule real-time sequencing[18].Resistance genes were analyzed using ResFinder 2.1,and mobile elements were determined using bioinformatics tools provided by IS Finder.The entireL.adecarboxylata17YN198 genome sequence was deposited in the GenBank database under the accession number CP106959-CP106963.L.adecarboxylata17YN198 contained a single circular chromosome with a length of 4,725,550 base pairs(bp) and a GC content of 55.69%.The chromosome carried 4,305 protein-coding genes,86 tRNA genes, and 25 rRNA genes (Figure 1).

Figure 1.Circular map of the L.adecarboxylata 17YN198 genome was designed using CGView.The gene marked in red in the outermost circle is the drug resistance gene obtained from card database comparison.The outer ring denotes the ORFs on the positive strand.The next ring illustrates ORFs on the complementary strand.The black circle presents GC content.The inner rings show G+C content and G+C skew,where peaks represent the positive (outward) and negative (inward) deviation from the mean G+C content and G+C skew,respectively.

Analysis of acquired resistance genes using ResFinder 2.1 showed thatL.adecarboxylata17YN198 harbored 33 antimicrobial resistance genes encoding resistance to tetracyclines (tetAandtetR),carbapenems (blaIMP),aminoglycosides [aph(3')-Ia,aadA6,(AGly)aacA4,aadA1],fluoroquinolones(qnrB5),and folate pathway antagonists (sul1)(Table 2).According to the assembly results,theL.adecarboxylata17YN198 isolate carried four plasmids.These included the 42,504-bpsul1-bearing plasmid pIMP-1 (CP106960),155,030-bp plasmid 2(CP106961),115,001-bpblaIMP-harboring plasmid 3(CP106962),and 52,474-bp plasmid 4 (CP106963).Plasmids 2 and 4 did not harbor any antimicrobial resistance genes.The results showed a susceptibility pattern consistent with the presence ofblaIMPandsul1,that is,resistance to meropenem,ertapenem,and trimethoprim-sulfamethoxazole.TheblaIMPgene harbored in the strain was most similar toblaIMP-79at the nucleotide level,with a similarity of 99.40%(737/741).The carbapenemase IMP encoded by theblaIMPgene had 100% amino acid identity with carbapenemase IMP-1.

TheblaIMP-harboring plasmid,designated as plasmid 3,had a length of 42,504 bp and an average GC content of 51.70%.Plasmid 3 belonged to the IncN3-incompatible group and contained three resistance genes [blaIMP-1,aadA6,and (AGly)aacA4](Figure 2).Plasmid comparison using BLASTn and the NCBI plasmid database (ftp.ncbi.nlm.nih.gov:/refseq/release/plasmid/) revealed that plasmid 3 is a novel plasmid.Although plasmid 3 did not carry ISs elements,it carried a type IV secretion system (virB1,virB3–virB6,andvirB8–virB11).This suggests that the transfer of resistance genes may be related to T4SS binding.SinceL.adecarboxylata17YN198 was isolated from a newborn female,blaIMP-1-harboring plasmid 3 may represent a newly emerging risk factor for the spread of carbapenemase resistance.

Figure 2.Schematic map of plasmid 3.Genes are denoted by arrows and colored based on gene function classification.The innermost circle presents GC-Skew (G-C/G+C) with a window size of 1,000 and step size of 500.The black circle presents GC content.Backbone and accessory module regions are also shown.

Infections caused byL.adecarboxylatahave likely been underestimated for several decades because of the difficulty in correctly identifying the bacterium,leading to underreporting in the medical literature[19].L.adecarboxylatawas previously considered an opportunistic pathogen.However,this may be due to misdiagnosis because this organism shares several biochemical characteristics withEscherichia coli.L.adecarboxylatais now considered a pathogenic organism due,in part,to the use of modern identification techniques such as API 20E(bioMérieux,Craponne,France) and MALDI-TOF MS,which have been used to accurately identify and isolateL.adecarboxylatafromE.coli.

To the best of our knowledge,this is the first report of carbapenem-resistantL.adecarboxylataisolated from a healthy newborn.TheL.adecarboxylatastrain isolated in this study carried four plasmids that may serve as reservoirs for antibiotic resistance genes.This case highlights the importance of consideringL.adecarboxylataas a potential cause of infections in children.These findings suggest that close monitoring of resistant strains in the human gut microbiota should become routine clinical practice to prevent the occurrence of infections.

Data Availability StatementThe datasets generated in this study are available in GenBank:SAMN31079621 and CP106959-CP106963.

#Correspondence should be addressed to LI Juan,Email: lijuan@icdc.cn

Biographical note of the first author: MENG Shuang,female,born in 1981,Doctor of Clinical Medicine,majoring in clinical testing diagnostics and bacterial resistance.