Molecular characterization of metallo-ß-lactamase- producing carbapenem-resistant Enterobacter cloacae complex isolated in Heilongjiang Province of China.

BACKGROUND: Enterobacter cloacae complex (ECC) is one of the most common extended-spectrum ß-lactamase and carbapenemase-producing pathogen that threatens millions of the elderly and vulnerable sick persons. The objective of this study was to perform the molecular characteristics of the carbapenem-resistant E. cloacae complex (CREC) emerged in Heilongjiang Province of China. METHODS: Six CREC strains were isolated from the patients with infectious diseases. The identities of ECC isolates were confirmed by sequencing the polymerase chain reaction (PCR) products of 16S rRNA gene. The characterization of the CREC isolates were analyzed by sequencing PCR products of the carbapenemase, ampC and fluoroquinolone resistance genes and performing multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and whole genome sequencing. RESULTS: All 6 isolates harbored multiple resistance genes. Of them, 5 carried metallo-ß-lactamases and one was blaKPC-2-positive. The levofloxacin and ciprofloxacin-resistant strains had substitutions of gyrA83, gyrA87, and parC80 in the quinolone-resistance determining regions. The MLST analyses revealed that 6 isolates belonged to five sequence types (ST520, ST528, ST1119, ST1120, and ST93) while the PFGE patterns of the isolates fallen into four clusters. The strain ST1120 was found to carry two separated plasmids that encode blaNDM-1 and blaIMP-4. CONCLUSIONS: Our study, for the first time, identified a CREC strain that co-produces blaNDM-1 and blaIMP-4 in the Northeast China. Our finding emphasizes an urgent need for more intensive surveillance and precaution measures to prevent the CERC spread.

Correction to: Outbreak of carbapenem-resistant Acinetobacter baumannii carrying the carbapenemase OXA-23 in ICU of the eastern Heilongjiang Province, China.

After publication of the original article [1], we were notified that an author's name has been incorrectly spelled. Sedzro Divine Mensal should be replaced with Sedzro Divine Mensah.

Outbreak of carbapenem-resistant Acinetobacter baumannii carrying the carbapenemase OXA-23 in ICU of the eastern Heilongjiang Province, China.

BACKGROUND: To investigate the carbapenem resistance mechanisms and clonal relationship of carbapenem-resistant Acinetobacter baumannii (CRAB) strains isolated in the intensive care unit (ICU) of the First Affiliated Hospital of Jiamusi University, management approaches to ICU clonal CRAB outbreaks were described. METHODS: The sensitivity of the antibiotic was determined using the VITEK-2 automated system. Carbapenemase genes (blaTEM, blaSHV, blaKPC, blaNDM, blaIMP-4, blaVIM, blaOXA-23, blaOXA-24, blaOXA-51, and blaOXA-58), AmpC enzyme genes (blaACC, blaDHA, blaADC), and ISAba1 were assessed for all collected isolates using polymerase chain reaction (PCR). The transfer of resistance genes was investigated via conjugation experiments. The clonal relationship of isolates was determined via enterobacterial repetitive intergenic consensus (ERIC)-PCR and multilocus sequence typing (MLST). When the detection rate of CRAB increased from 25% in 2010 to 92% in 2014, a number of actions were initiated, including enhanced infection control, staff education, and the cleaning of the hospital environment. RESULTS: Clinical isolates were positive for the following genes: blaOXA23, blaOXA51, blaOXA24, blaADC, blaTEM, ISAba1, ISA-23, and ISA-ADC; however, blaOXA58, ISA-51, blaNDM, blaIMP, blaKPC, blaTEM, blaSHV, blaVIM, and blaACC were not detected. Four carbapenem-resistant isolates successfully transferred plasmids from A. baumannii isolates to E. coli J53. MLST showed that all strains belonged to ST2 except for one isolate, which belonged to the new genotype ST1199. The ERIC-PCR method found the following three genotypes: type A in 8, type B in 12, type C in 1, and two profiles (A, B) belonged to ST2. After taking control measures, the prevalence of CRAB isolates decreased, and the discovery rate of CRAB dropped to 11.4% in 2017. CONCLUSION: The obtained result suggests that blaOXA-23-producing CC2 isolates were prevalent in the ICU of the First Affiliated Hospital of Jiamusi University. Targeted surveillance was implemented to identify the current situation of the ICU and the further implementation of infection control effectively prevented the spread of nosocomial infection.

Mechanism for transmission and pathogenesis of carbapenem-resistant Enterobacterales harboring the carbapenemase IMP and clinical countermeasures.

Carbapenem-resistant Enterobacterales (CRE) are some of the most important pathogens causing infections, which can be challenging to treat. We identified four blaIMP-carrying CRE isolates and collected clinical data. The transferability and stability of the plasmid were verified by conjugation, successive passaging, and plasmid elimination assays. The IncC blaIMP-4-carrying pIMP4-ECL42 plasmid was successfully transferred into the recipient strain, and the high expression of traD may have facilitated the conjugation transfer of the plasmid. Interestingly, the plasmid showed strong stability in clinical isolates. Whole-genome sequencing was performed on all isolates. We assessed the sequence similarity of blaIMP -harboring plasmid from our institution and compared it to plasmids for which sequence data are publicly available. We found that four blaIMP-carrying CRE belonged to four different sequence types. The checkerboard technique and time-kill assays were used to investigate the best antimicrobial therapies for blaIMP-carrying CRE. The time-kill assay showed that the imipenem of 1× minimum inhibitory concentration (MIC) alone had the bactericidal or bacteriostatic effect against IMP-producing strains at 4-12 h in vitro. Moreover, the combination of tigecycline (0.5/1/2 × MIC) and imipenem (0.5/1 × MIC) showed a bactericidal effect against the blaIMP-26-carrying CRECL60 strain.IMPORTANCECarbapenem-resistant Enterobacterales (CRE) are an urgent public health threat, and infections caused by these microorganisms are often associated with high mortality and limited treatment options. This study aimed to determine the clinical features, molecular characteristics, and plasmid transmissible mechanisms of blaIMP carriage as well as to provide a potential treatment option. Here, we demonstrated that conjugated transfer of the IncC blaIMP-4-carrying plasmid promotes plasmid stability, so inhibition of conjugated transfer and enhanced plasmid loss may be potential ways to suppress the persistence of this plasmid. The imipenem alone or tigecycline-imipenem combination showed a good bactericidal effect against IMP-producing strains. In particular, our study revealed that imipenem alone or tigecycline-imipenem combination may be a potential therapeutic option for patients who are infected with IMP-producing strains. Our study supports further trials of appropriate antibiotics to determine optimal treatment and emphasizes the importance of continued monitoring of IMP-producing strains in the future.

Molecular characterization of NDM-1-producing carbapenem-resistant E. cloacae complex from a tertiary hospital in Chongqing, China.

Background: The aim of this study was to clarify the molecular characterization of NDM-1-producing carbapenem-resistant Enterobacter cloacae complex (CREL) at a teaching hospital in Chongqing, China. Methods: Antimicrobial susceptibility and resistance genes were analyzed. Epidemiological relationship was analyzed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Conjugation experiments were performed to determine the transferability of plasmids. Whole-genome sequencing (WGS) of strains was implemented, and the genetic environment of the bla NDM-1- and mcr-9-carrying plasmids was analyzed. Results: A total of 10 bla NDM-1-positive CREL isolates were identified. All isolates harbored multiple resistance genes. ECL68 and ECL78 co-produce bla NDM-1 and mcr-9. Among the four different sequence types (STs) detected, ST1466 was assigned as a novel ST. Six isolates exhibited highly similar PFGE patterns. Conjugation assay proved that all plasmids containing bla NDM-1 or mcr-9 could be transferred to the recipient Escherichia coli. WGS indicated that bla NDM-1 genes were carried by diverse plasmids, including IncHI2/IncN, IncX3, and one unclassified plasmid type. The backbone structure of these plasmids is involved in replication initiation (repAB), partitioning (parABM), and conjugation/type IV secretion (tra/virB). Analysis of the genetic environment showed that bla NDM-1 in three plasmids exhibited a highly similar structure to protype Tn125. Co-existence of bla NDM-1 and the colistin resistance gene mcr-9 was detected in the two isolates, ECL68 and ECL78. In ECL68, bla NDM-1 and mcr-9 were present on the same plasmid while located in two separate plasmids in ECL78. The genetic environment of mcr-9 was organized as IS26-wbuC-mcr-9-IS903-pcoS-pcoE-rcnA-rcnR, and the two-component system encoding genes qseC and qseB was not found in two plasmids, which could explain mcr-9-harboring strains' colistin susceptibility. Conclusions: We first report a nosocomial outbreak of NDM-1-producing E. cloacae complex ST177 in China. Conjugative plasmids contributed to the horizontal transfer of antibiotic resistance genes. The prevalence and even coexistence of bla NDM-1 and mcr-9 may further threaten public health. Our results highlight further surveillance for bla NDM-1, and mcr-9 is essential to prevent its dissemination.

Molecular antibiotic resistance mechanisms and co-transmission of the mcr-9 and metallo-ß-lactamase genes in carbapenem-resistant Enterobacter cloacae complex.

Carbapenem-resistant Enterobacter cloacae complex (CRECC) has increasingly emerged as a major cause of healthcare-associated infections, with colistin being one of the last-resort antibiotics of treatment. Mobile colistin resistance (mcr)-9 is a member of a growing family of mcr genes and has been reported to be an inducible gene encoding an acquired phosphoethanolamine transferase. Here, we collected 24 ECC strains from Chongqing, China from 2018 to 2021. Subsequently, antibiotic resistance genes and the transmission dynamics of the strains were determined by PCR, whole-genome sequencing, and bioinformatic analysis. The mcr-9 was identified in IncHI2/2A or IncHI2/2A + IncN plasmids from six CRECC strains and was co-located with bla NDM-1 or bla IMP-4 in 2/6 plasmids. The genetic environment of mcr-9.1 was composed of IS903B-mcr-9.1-wbuC-IS26 in the five mcr-9.1-harboring-plasmid, but IS1B was located downstream of mcr-9.2 in the pECL414-1 sequence. We also found that the pNDM-068001 plasmid carrying mcr-9.1 could be a hybrid plasmid, formed by a Tn6360-like bla NDM-1 region inserted into an mcr-9.1-positive IncHI2/2A plasmid. A conjugation assay showed that plasmids mediated the co-dissemination of mcr-9 and metallo-ß-lactamase (MBL) genes. In addition, we performed induction assays with sub-inhibitory concentrations of colistin and found an increase in the relative expression levels of the mcr-9.2, qseC, and qseB genes, as well as an increase in the minimum inhibitory concentration values of colistin in the CRECC414 strain. These findings provide a basis for studying the regulatory mechanisms of mcr-9 expression and highlight the importance of effective monitoring to assess the prevalence of MBL and mcr-9 co-existing plasmids.

An Outbreak of Carbapenem-Resistant Klebsiella pneumoniae in an Intensive Care Unit of a Major Teaching Hospital in Chongqing, China.

Background: Due to the critical condition and poor immunity of patients, the intensive care unit (ICU) has always been the main hospital source of multidrug-resistant bacteria. In recent years, with the large-scale use of antibiotics, the detection rate and mortality of carbapenem-resistant Klebsiella pneumoniae (CRKP) have gradually increased. This study explores the molecular characteristics and prevalence of CRKP isolated from the ICU ward of a tertiary hospital in China. Methods: A total of 51 non-duplicated CRKP samples isolated from the ICU were collected from July 2018-July 2020. The enzyme production of the strains was preliminarily screened by carbapenemase phenotypic test, and drug-resistant and virulence genes were detected by PCR. The transferability of plasmid was verified by conjugation test. The minimal inhibitory concentration (MIC) was determined by microbroth dilution method and genetic diversity was detected by multilocus sequence typing and pulsed-field gel electrophoresis. Results: blaKPC-2 was the only carbapenemase detected. The major virulence genes were uge (100%), mrkD (94.1%), kpn (94.1%), and fim-H (72.5%), while wcag, ironB, alls and magA genes were not detected. One sequence type ST1373 strain, hypervirulent K. pneumoniae (hvKP), was detected. CRKP strains were highly resistant to quinolones, cephalosporins, aminoglycosides, and polymyxin, but susceptive to tigecycline and ceftazidime-avibactam. The success rate of conjugation was 12.2%, indicating the horizontal transfer of blaKPC-2 . Homology analysis showed that there was a clonal transmission of ST11 CRKP in the ICU of our hospital. Conclusion: The present study showed the outbreak and dissemination in ICU were caused by ST11 CRKP, which were KPC-2 producers, and simultaneously, also carried some virulence genes. ST11 CRKP persisted in the ward for a long time and spread among different areas. Due to the widespread dispersal of the transferable blaKPC-2 plasmid, the hospital should promptly adopt effective surveillance and strict infection control strategies to prevent the further spread of CRKP. Ceftazidime-avibactam showed high effectiveness against CRKP and could be used for the treatment of ICU infections.

Carbapenemase Production and Epidemiological Characteristics of Carbapenem-Resistant Klebsiella pneumoniae in Western Chongqing, China.

Background: This study aimed to determine the molecular characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates in a hospital in western Chongqing, southwestern China. Methods: A total of 127 unique CRKP isolates were collected from the Yongchuan Hospital of Chongqing Medical University, identified using a VITEK-2 compact system, and subjected to microbroth dilution to determine the minimal inhibitory concentration. Enterobacteriaceae intergenic repeat consensus polymerase chain reaction and multilocus sequence typing were used to analyze the homology among the isolates. Genetic information, including resistance and virulence genes, was assessed using polymerase chain reaction. The genomic features of the CRKP carrying gene blaKPC-2 were detected using whole-genome sequencing. Results: ST11 was the dominant sequence type in the homology comparison. The resistance rate to ceftazidime-avibactam in children was much higher than that in adults as was the detection rate of the resistance gene blaNDM (p < 0.0001). Virulence genes such as mrkD (97.6%), uge (96.9%), kpn (96.9%), and fim-H (84.3%) had high detection rates. IncF (57.5%) was the major replicon plasmid detected, and sequencing showed that the CRKP063 genome contained two plasmids. The plasmid carrying blaKPC-2, which mediates carbapenem resistance, was located on the 359,625 base pair plasmid IncFII, together with virulence factors, plasmid replication protein (rep B), stabilizing protein (par A), and type IV secretion system (T4SS) proteins that mediate plasmid conjugation transfer. Conclusion: Our study aids in understanding the prevalence of CRKP in this hospital and the significant differences between children and adults, thus providing new ideas for clinical empirical use of antibiotics.

Microbial Characteristics and Genomic Analysis of an ST11 Carbapenem-Resistant Klebsiella pneumoniae Strain Carrying bla KPC-2 Conjugative Drug-Resistant Plasmid.

BACKGROUND: The sequence type 11 (ST11) carbapenem-resistant Klebsiella pneumoniae (CRKP) carrying bla KPC-2 has been widespread all over the world, and it has been reported frequently in China. The bla KPC-2 located on the mobile genetic element brings tremendous pressure to control the spread and outbreak of resistant bacteria. Whole-genome sequencing (WGS) technology can comprehensively and in-depth display the molecular characteristics of drug-resistant bacteria, providing a basis for evaluating the genetic diversity within the CRKP genome. METHODS: The ST11 CRKP in this study was collected in the intensive care unit of a major teaching hospital. PCR and Sanger sequencing confirmed the existence of bla KPC-2. The AST-GN card and the microbroth dilution test were used for antimicrobial susceptibility testing. The transferability of plasmid was verified by a conjugation test. The whole genome is sequenced using the Illumina HiSeq short-read and Oxford Nanopore long-read sequencing technology. RESULTS: The studied strain was named CRKP63, which is a multi-drug resistance bacteria, which carries bla KPC-2 and bla SHV-182. Its genome consists of a circular chromosome of 5,374,207 bp and an IncFII plasmid named pKPC-063001 of 359,625 bp. In the drug-resistant plasmid pKPC-063001, the key carbapenem resistance gene bla KPC-2 was located in the genetic context with insertion sequence ISKpn27 upstream and ISKpn6 downstream and bracketed by IS26. The three copies of the IS26-ISKpn27-bla KPC-2-ISKpn6-IS26 unit were present in tandem. bla KPC-2 can be transferred horizontally between other species by conjugation, the complete type IV secretion system (T4SS) structure helps to improve the adaptability of bacteria to the external environment, strengthen the existence of drug-resistant bacteria, and accelerate the spread of drug resistance. CONCLUSION: High-throughput sequencing has discovered the different surrounding environments of bla KPC-2, which provides a new idea for further revealing the transmission and inheritance of bla KPC-2 at the molecular level. In order to control the further spread and prevalence of drug-resistant bacteria, we should pay close attention to the changes in the genetic environment of bla KPC-2 and further study the transcription and expression of T4SS.

Antibiotic susceptibility and molecular analyses of clinical Enterobacter cloacae isolates in Eastern Heilongjiang Province, China.

BACKGROUND: Enterobacter cloacae is an emerging opportunistic pathogen. We retrospectively conducted a study to assess antimicrobial susceptibility and investigated the Molecular characteristics of carbapenem-resistant Enterobacter cloacae (CREL) isolates. METHODS: Three hundred forty-two isolates of Enterobacter cloacae were collected from January 2014 to December 2018. Ten strains of CREL were collected for further research. The species identifications and minimum inhibitory concentrations (MICs) of all antibiotics tested were analyzed using the Vitek 2 Compact system (BioMerieux, France) and supplemented by the disk diffusion method. Polymerase chain reaction (PCR) was performed to detect extended-spectrum ß-lactamase (ESBL) and carbapenemase resistance genes. RESULTS: The results showed that most of the isolates remained susceptible to tested antibiotics; however, the resistance rate of Cefepime has been increasing in recent years. One strain co-producing New Delhi Metallo-ß-lactamase NDM-1 and Imipenem hydrolase IMP-4. NDM-1 and IMP-4-producing isolates highlight that active surveillance is necessary to prevent the further spread of the bacteria. Multilocus sequence typing (MLST) showed that two KPC-producing isolates assigned to ST93, two isolates carrying NDM-1 assigned to ST1120. Moreover, the MEGA analysis showed that ST93, ST256, and ST1120 have homology, showing that CREL in our area has a potential spread risk. CONCLUSIONS: These findings indicating that CREL clonal dissemination may occurred in this region and should be taken seriously concern. Our study highlights an urgent need to monitor these isolates to prevent their further spread.

The in vitro activity of polymyxin B and tigecycline alone and combination with other antibiotics against carbapenem-resistant Enterobacter cloacae complex isolates, including high-risk clones.

BACKGROUND: The emergence of carbapenem-resistant Enterobacteriaceae (CRE) has become a significant problem for global public health. Currently, treatments program is minimal. This study aimed to evaluate the molecular mechanisms of carbapenem-resistant Enterobacter cloacae complex isolates (CREC) infections. Methods: Resistance genes were detected using PCR with specific primers. Multilocus sequence typing (MLST) was also performed. Furthermore, we evaluated the effects of polymyxin B (PMB) and tigecycline (TGC) antibiotics (Abs) alone and in combination with meropenem (MEM), amikacin (AMK), and levofloxacin (LEV) against CREC isolates. The results were then compared with in vitro synergy testing results obtained from time-kill assays (TKAs), and the microdilution checkerboard method. RESULTS: The synergistic efficiency of PMB + TGC was also evaluated. Abs use clinically achievable concentrations to determine the antibacterial effects of the Ab. Similar sequence type (ST) classifications had a comparably resistant phenotype; PMB-based combination therapy is better than TGC-based combination therapy. CONCLUSIONS: we found that the combination of PMB + AMK is promising for the treatment of AMK-sensitive CREC. The high-risk ST93 carrying the bla KPC-2 gene should be monitored.