BackgroundIn recent years, New Delhi metallo-beta-lactamases 1 (bla NDM-1) has been reported with increasing frequency and become prevalent. The present study was undertaken to investigate the epidemiological dissemination of the bla NDM-1 gene in Enterobacter cloacae isolates at a teaching hospital in Yunnan, China.MethodsAntimicrobial susceptibility testing was performed using VITEK 2 system and E test gradient strips. The presence of integrons and insertion sequence common region 1 were examined by PCR and sequencing. Clonal relatedness was assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. Conjugation experiments and Southern blot hybridization were performed to determine the transferability of plasmids.ResultsTen E. cloacae isolates and their Escherichia coli transconjugants were exhibited similar resistant patterns to carbapenems, cephalosporins and penicillins. 8 (80%) of E. cloacae isolates carried class 1 integron and 1 (12.5%) carried class 2 integron. Integron variable regions harbored the genes which encoded resistance to aminoglycosides (aadA1, aadA2, aadA5, aadB, aac(6′)-Ib-cr), sulfamethoxazole/trimethoprim (dfrA17, dfrA12, dfrA15) and Streptozotocin (sat2). Six E. cloacae isolates belonged to ST74 and exhibited highly similar PFGE patterns. Each isolate shared an identical plasmid with ~33.3 kb size that carried the bla NDM-1 gene, except T3 strain, of which the bla NDM-1 gene was located on a ~50 kb plasmid.ConclusionsOur findings suggested that plasmid was able to contribute to the dissemination of bla NDM-1. Hence, more attention should be devoted to monitor the dissemination of the bla NDM-1 gene due to its horizontal transfer via plasmid. In addition, nosocomial surveillance system should actively monitor the potential endemic clone of ST74 to prevent their further spread.

Background: New Delhi metallo-beta-lactamase 1 (NDM-1) is considered to be an important factor of antimicrobial resistance in Enterobacteriaceae. In China, the blaNDM-1 gene has been mostly detected in carbapenem-resistant Acinetobacter spp. but is less reported in Enterobacteriaceae and more rarely found in E. cloacae. Objectives: This study explored the genetic features of the blaNDM-1 gene of E. cloacae and a blaNDM-1knockout mutant was constructed using Red homologous recombination. In addition, the effect of the knockout on antimicrobial resistance, growth ability, and in vitro competitiveness was investigated. Methods: The upstream and downstream structures of the blaNDM-1 gene were analyzed in ten E. cloacae isolates using primer walking and PCR mapping. A blaNDM-1 knockout mutant was constructed through Red homologous recombination and verified by PCR, RT-qPCR, and sequencing. The antimicrobial susceptibility, growth curves, and in vitro growth competitiveness were compared between the blaNDM-1 knockout mutant and the parental strain. Results: All E. cloacae study isolates except for strain T10, contained an identical blaNDM-1 gene structure. The ∆ISAba125 truncated by ISEc33 element and the bleo followed by a ∆trpF and ISSen4 was located immediately upstream and downstream of T1-T9 strains. However, the ∆ISAba125 and the bleo followed by a ∆trpF were located immediately upstream and downstream, respectively, in the T10 strain. PCR, RT-qPCR, and DNA sequencing analyses showed that the blaNDM-1 knockout mutant was successfully constructed. The blaNDM-1 knockout mutant and the parental strain exhibited similar resistance patterns to penicillin, cephalosporins, aminoglycosides, tetracycline, and quinolones. Both strains displayed similar growth curves in Luria Broth. The competition index (CI), defined as the knockout mutant/parental strain ratio was 0.69 in the competition experiment in vitro. Conclusions: The DNA regions upstream and downstream of the blaNDM-1 gene often contained insertion sequences and elements. Red homologous recombination was successfully used to knock out blaNDM-1 in E. cloacae, which allowed us to decipher the links between this gene, antimicrobial resistance, and bacterial growth competitiveness.

Background: Carbapenem-resistant Enterobacteriaceae (CRE) has become a public health threat due to resistance to multiple antibiotics. The production of β-lactamase is the most important resistance mechanism of Enterobacteriaceae. Although isolates producing KPC-2 or NDM-1 enzymes have been reported widely, isolates co-producing KPC-2, NDM-1, TEM-1, TEM-95, SHV-66, and other β-lactamases have rarely been detected in the same strain, especially in Enterobacter cloacae. Objectives: In this study, we identified and sequenced the genome of carbapenem-resistant E. cloacae ECL189 to in-depth analyze the resistance and transmission mechanisms of E. cloacae. Methods: We investigated the antimicrobial susceptibility of ECL189 by a VITEK 2 system, E-test gradient strips, and K-B method. Whole-genome sequencing was used by the PacBio RS II platform and Illumina HiSeq 4000 platform. Antimicrobial resistance genes, virulence genes, non-coding RNA, and repeat sequences were predicted by biological information databases. A PCR was used to further confirm that the blaKPC-2, blaNDM-1, blaTEM-1, blaTEM-95, and blaSHV-66 genes existed in ECL189. A conjugation experiment was performed to determine the transferability of resistance. Molecular typing of ECL189 was done by multilocus sequence typing (MLST). Results: Enterobacter cloacae ECL189 was resistant to 21 out of 23 tested antibiotics, but its transconjugant was resistant to 10 out of 18 tested antibiotics. The genome of ECL189 consisted of a 5,026,406 bp chromosome and four circular plasmids. In total, 26 resistance genes and 58 resistance proteins were identified. In addition, 77 determinants associated with bacterial virulence were identified. A large number of resistance and virulence genes were located in the plasmids. The results of whole-genome sequencing were consistent with the β-lactamase genes. The MLST analysis revealed that this strain belonged to ST74. Conclusions: This study further revealed the resistance, virulence, and transmission mechanisms of carbapenem-resistant E. cloacae. Resistance and virulence genes spread in bacteria by the horizontal transfer of plasmids, which should attract more attention in relevant departments.

Introduction. The bla NDM-1 -positive Enterobacter cloacae has led to limited therapeutic options for clinical treatment. Hypothesis/Gap Statement. Analysing the antimicrobial resistance and molecular typing of bla NDM-1-positive E. cloacae is of great significance. Meanwhile, the effect of the bla NDM-1 gene on the virulence and pathogenicity of E. cloacae remains unclear and should be assessed. Aim. To understand bla NDM-1-positive E. cloacae from different perspectives. Methodology. The PCR was used to screen bla NDM-1-positive E. cloacae , then, antimicrobial susceptibility tests and multilocus sequence typing (MLST) were performed on them; sixty-nine strains of bla NDM-1-negative E. cloacae were collected as the controls, 28 pairs of virulence-related genes' carriage and biofilm-forming ability were detected for preliminary evaluation of the virulence phenotype of the strains; to gain insight into the effect of the bla NDM-1 gene on the virulence and pathogenicity of E. cloacae , the bla NDM-1-positive E. cloacae T2 (NDM-1), the T2 bla NDM-1 knockout strain (ΔNDM-1) and ATCC13047 (ST) were studied, compared the motility, anti-serum killing ability, and virulence to cells. Then, the mice intraperitoneal infection model was established, the survival curve, histopathological characteristics, bacterial load in spleen and the contents of cytokines were compared. Results. (1) Thirty-five bla NDM-1-positive E. cloacae exhibited multidrug resistance. MLST distinguished 12 STs, ST74 was the most common clonal type (11/35), followed by ST114 (10/35). (2) The detection rates of virulence genes clpB, icmf, VasD/Lip and acrA in the bla NDM-1-positive E. cloacae were significantly higher than those in bla NDM-1-negative E. cloacae (P<0.05), while there was no significant difference in the amount of biofilm formation between two groups. (3) The presence of bla NDM-1 gene attenuated the motility diameter of E. cloacae , but had no significant effect on their ability to resist serum killing, and the virulence to cells. The survival rate, histopathological changes, bacterial load in spleen and inflammatory cytokines were not significantly affected. Conclusions. (1) The bla NDM-1-positive E. cloacae exhibited multidrug resistance, and the MLST typing was mainly ST74 and ST114, with a small-scale clonal spread of the ST114 strain in the hospital NICU ward. (2) The bla NDM-1 gene did not affect the virulence and pathogenicity of E. cloacae .