Carbapenem Resistance Genes Research Articles

Genome Analysis of Klebsiella pneumoniae Reveals International High-Risk Pandemic MDR Clones Emerging in Tertiary Healthcare Settings in Uganda.

Klebsiella pneumoniae is a threat to public health due to its continued evolution. In this study, we investigated the evolution, convergence, and transmission of hypervirulent and multi-drug resistant (MDR) clones of K. pneumoniae within healthcare facilities in Uganda. There was high resistance to piperacillin (90.91%), cefuroxime (86.96%), ceftazidime (84.62%), cefotaxime (84.00%), amoxicillin/clavulanate (75%), nalidixic acid (73.68%), and nitrofurantoin (71.43%) antibiotics among K. pneumoniae isolates. The isolates were genetically diverse, consisting of 20 different sequence types (STs) and 34 K-serotype groups. Chromosomal fosA (for fosfomycin) and oqxAB efflux pump genes were detected in all isolates. Two carbapenem resistance genes, blaNDM-5 and blaOXA-181 plus extended-spectrum beta-lactamase (blaCTX-M-15) gene (68.12%), quinolone-resistant genes qnrS1 (28.99%), qnrB1 (13.04%), and qnrB6 (13.04%) and others were found. All, except three of the isolates, harbored plasmids. While the isolates carried a repertoire of virulence genes, only two isolates carried hypervirulent genes demonstrating a low prevalence (2.90%) of hypervirulent strains. Our study demonstrated genetically diverse populations of K. pneumoniae, low levels of carbapenem resistance among the isolates, and no convergence of MDR and hypervirulence. Emerging high-risk international pandemic clones (ST11, ST14, ST147, ST 86 and ST307) were detected in these healthcare settings which are difficult to treat.

Molecular characterization of IncFII plasmid carrying blaNDM-5 in a Salmonella enterica serovar Typhimurium ST34 clinical isolate in China.

In this study, an IncFII plasmid pIncFII-NDM5 carrying blaNDM-5 was found in carbapenem-resistant Salmonella enterica serovar Typhimurium (S. enterica serovar Typhimurium), which has conjugative transferability and carried blaNDM-5, bleMBL, mph(A), and blaTEM-1 four resistance genes that can mediate resistance to multiple antibiotics including cephalosporins, beta-lactamase inhibitor combinations, carbapenems, and macrolides. Phylogenetic analysis showed that 1104-65 and 1104-75 were closely related to other S. enterica serovar Typhimurium in this area. The above-mentioned S. enterica serovar Typhimurium chromosome carries blaCTX-M-55, qnrS1, and tet(A) genes, so the antibiotic resistance of isolates will be further enhanced after obtaining the pIncFII_NDM5-like plasmid. Meanwhile, we discovered a novel genetic structure of blaNDM-5 mediated by the IS26 composite transposon, which will expand our understanding of the emergence and spread of carbapenem-resistance genes. Altogether, the presence of the IncFII plasmid pIncFII-NDM5 further underscores the need for vigilant surveillance and appropriate infection control measures to mitigate the impact of carbapenem-resistant S. enterica serovar Typhimurium in clinical settings.

Clinical and Molecular Characteristics of Carbapenemase-Producing E. coli Strains from Patients with Biliary System Diseases and Hematological Malignancies.

This study aims to investigate the clinical and molecular characteristics of carbapenemase-producing E. coli strains (CPECO). We collected 38 non-repetitive CPECO strains, identified them using MALDI-TOF, and assessed their antimicrobial susceptibility via the VITEK-Compact II system. We gathered demographic and clinical patient data. Phenotypic assays were employed to detect carbapenemase types. Polymerase chain reaction (PCR) was utilized to identify the carbapenemase genes. Seven housekeeping genes were amplified and sequenced to determine the multilocus sequence typings (MLSTs). These CPECO strains, primarily isolated from aseptic site and stool screening specimens, exhibited significant resistance to most clinical antibiotics, except for tigecycline and amikacin. Most patients had underlying medical conditions and underwent invasive procedures. There were significant differences among patients concerning the presence of malignancies, digestive system disorders, endoscopic retrograde cholangiopancreatography (ERCP) surgeries and abdominal drainage tubes. However, no significant differences were observed among patients regarding conditions, including hypertension, diabetes, respiratory diseases, urinary diseases and cardiovascular diseases, as well as invasive procedures such as deep venous catheterization, endotracheal intubation and gastrointestinal catheterization. Metallo-β-lactamase was primarily responsible for carbapenem resistance, including blaNDM-5(24/38), blaNDM-1(5/38), blaNDM-9(1/38) and blaIMP-4(1/38). Additionally, 7 CPECO strains carried blaKPC-2. The distribution of CPECO sequence types (STs) was diverse, with seven strains being ST131, six strains being ST410, three strains each of ST1196 and ST10, although most STs were represented by only one strain. CPECO infections in patients with biliary system diseases may result from intestinal CPECO translocation, with ERCP surgery potentially facilitating this. Meanwhile, malignant tumor was found to be a significant factor affecting CPECO infections in patients with hematological diseases. blaNDM-5, blaNDM-1 and blaNDM-9 were primarily responsible for carbapenem resistance in CPECO strains. The emergence of carbapenem-resistant ST131 and ST410 strains should be alert to prevent the spread of carbapenem-resistant genes within high-risk epidemic clones.

Nutritional analysis and characterization of carbapenemase producing-Klebsiella pneumoniae resistant genes associated with bovine mastitis infected cow's milk.

The current study was designed to analyze nutritional parameters and to characterize carbapenemase producing-Klebsiella pneumoniae isolates from bovine mastitic cow's milk. Out of 700 milk samples K. pneumoniae was identified by phenotypic and molecular techniques along with their antibiogram analysis and nutritional analysis was performed using the procedure of Association of Official Analytical Chemists. Carbapenemase-producing K. pneumoniae was detected by phenotypic CarbaNP test followed by molecular characterization of their associated resistant genes blaVIM, blaKPC, blaOXA-48, blaNDM, and blaIMP along with insertion sequence common region 1 (ISCR1) and integrons (Int1, Int2, and Int3) genes. Among nutritional parameters, fat content was observed (2.99%) followed by protein (2.78%), lactose (4.32%), and total solid (11.34%), respectively. The prevalence of K. pneumoniae among bovine mastitis was found 25.71%. Antibiogram analysis revealed that more effective antibiotics was ceftazidime (80%) followed by amikacin (72%), while highly resistant antibiotics was Fusidic acid (100%). Distribution of carbapenemase producer K. pneumoniae was found 44.4%. Among carbapenem resistant genes blaKPC was found 11.25%, blaVIM 2.75%, blaNDM 17.5%, and blaOXA-48 7.5%, while blaIMP gene was not detected. Furthermore, distribution of ISCR1 was found 40%, while integron 1 was found 61.2% followed by integron 2 (20%), and integron 3 (5%). In conclusion, the recent scenario of carbapenemase resistant K. pneumoniae isolates responsible for mastitis may affect not only the current treatment regime but also possess a serious threat to public health due to its food borne transmission and zoonotic potential.

Occurrence of high-risk clones of Klebsiella pneumoniae ST11, ST340 and ST855 carrying the blaKPC-2, blaNDM-1, blaNDM-5 and blaNDM-7 genes from colonized and infected patients in Brazil.

Determine which ST clones were carrying the blaKPC, blaNDM, blaVIM, blaIMP and blaGES genes and their variants in clinical isolates of multidrug resistant K. pneumoniae. Ten K. pneumoniae isolates were obtained from the colonized and infected patients in a public hospital in the city of Recife-PE, in northeastern Brazil, and were further analyzed. The detection of carbapenem resistance genes and the 7 housekeeping genes (for MLST detection) were done with PCR and sequencing. The blaKPC and blaNDM genes were detected concomitantly in all isolates, with variants being detected blaNDM-1, blaNDM-5, blaNDM-7 and blaKPC-2. The blaKPC-2 and blaNDM-1 combination being the most frequent. Molecular typing by MLST detected three types of high-risk ST clones, associated with the clonal complex 258, ST11/CC258 in eight isolates, and ST855/CC258 and ST340/CC258 in the other two isolates. These findings are worrying, as they have a negative impact on the scenario of antimicrobial resistance, and show the high genetic variability of K. pneumoniae and its ability to mutate resistance genes and risk of dissemination via different ST clones.

Clinical and molecular epidemiology of carbapenem-resistant Enterobacteriaceae in pediatric inpatients in South China.

Carbapenem-resistant Enterobacteriaceae (CRE) are a substantial health burden. Existing management guidelines are primarily based on clonal evidence. Therefore, epidemiological data are required. We evaluated the risk factors for CRE colonization and described the molecular and clinical characteristics of CRE colonization in pediatric inpatients in Guangzhou, China. Pediatric inpatients from three hospitals in Guangzhou, China, between July 2019 and January 2021 were included. Fecal samples were collected and screened for CRE. Microbiological cultures were grown on MacConkey agar plates, and bacteria were identified with a mass spectrometry microbial identification system. Antimicrobial susceptibility was tested with a VITEK2 system, and carbapenemase genes were amplified. A case-control study was conducted to evaluate the risk factors for fecal CRE colonization in pediatric inpatients. Of 4,033 patients screened, 158 (3.92%) were positive for CRE, including Escherichia coli (51.27%), Klebsiella pneumoniae (37.97%), and Enterobacter cloacae (6.96%). The most common carbapenemase genes were blaNDM-5 (51.89%), blaNDM-1 (15.19%), and blaIMP-4 (7.60%) and blaKPC-2 (3.80%). Hematological malignancies, respiratory diseases, otolaryngological diseases, nervous system diseases, oral administration of third-generation cephalosporins, and the combined use of two or more antibiotics were independently associated with CRE colonization. The most prevalent carbapenem-resistant K. pneumoniae sequence types (STs) were ST11 (8.06%), ST37 (8.06%), and ST76 (8.06%), and the most prevalent carbapenem-resistant E. coli STs were ST10 (9.88%), ST48 (13.58%), and ST58 (8.64%). Pediatric inpatients were colonized by a diversity of CRE strains. Infection control and prevention measures should be implemented to reduce CRE colonization. IMPORTANCE This study assessed the clinical and molecular epidemiology of carbapenem-resistant Enterobacteriaceae in pediatric inpatients at three hospitals in South China by means of screening stool samples for carbapenem-resistant genes and a nested case-control study to determine risk factors for carriage of carbapenem-resistant Enterobacteriaceae. Of 4,033 fecal samples screened, 158 (3.92%) were positive for CRE, including Escherichia coli (51.27 %), Klebsiella pneumoniae (37.97%), and Enterobacter cloacae (6.96%). The most common carbapenemase genes harbored by gastrointestinal CRE strains were blaNDM-5, blaNDM-1, and blaIMP-4. Hematological malignancies, respiratory diseases, otolaryngological diseases, nervous system diseases, oral administration of third-generation cephalosporins, and the combined use of two or more antibiotics were independently associated with CRE colonization.

NDM-1- and OXA-23-producing Acinetobacter baumannii in wastewater of a Nigerian hospital.

Carbapenem-resistant Acinetobacter baumannii spp. are increasingly important pathogens with limited treatment options, and there is limited knowledge on the environmental factors contributing to their spread. We determined the occurrence of carbapenem-resistant A. baumannii in hospital wastewater and their phylogenetic relationships with clinical A. baumannii isolates. Grab samples of raw and treated hospital wastewater were collected monthly at the University College Hospital, Ibadan, Nigeria, between March 2021 and February 2022. Acinetobacter baumannii strains were selectively isolated and identified using VITEK2, and their whole genomes were sequenced on an Illumina platform. We performed antimicrobial susceptibility testing and in silico genomic characterization of the strains and determined their phylogenetic relationships to previously characterized clinical A. baumannii strains from Nigeria. A. baumannii complex isolates were recovered from wastewater throughout the study. Of the 82 isolates identified based on whole-genome sequences, 77 were A. baumannii. A. baumannii isolates had high resistance rates (≥48.1%) to 10 of 12 antimicrobials tested, and majority (42/77, 54.5%) were resistant to carbapenems, with bla NDM-1 being the most common (24/77, 31.2%) carbapenem resistance gene detected, followed by bla OXA-23 (n = 22, 28.6%). There was no statistically significant difference in carbapenem resistance rates or carbapenem gene carriage between the raw and treated wastewater isolates. Most of the isolates belonged to novel or sparsely described lineages, some of which were closely related to clinical isolates. The release of inadequately treated hospital wastewater into the environment may contribute to the increased spread of carbapenem-resistant and clinically important A. baumannii lineages in Ibadan, Nigeria. IMPORTANCE Acinetobacter baumannii is a leading cause of hospital-associated infections globally. A. baumannii reservoirs outside hospital settings are still unknown, and their occurrence in the environment is linked to clinical and anthropogenic activities. Although the risk of transmission of A. baumannii from environmental sources to humans is not fully understood, these sources pose significant risks for the continued dissemination of A. baumannii and their resistance traits. This study provides evidence that diverse and clinically relevant A. baumannii strains, many of which are resistant to carbapenems, are constantly being discharged into the environment through inadequately treated hospital wastewater. We further elucidate potential transmission routes between the environment and clinical infections and demonstrate the high prevalence of carbapenem resistance genes on highly mobile transposons among these strains. Our findings highlight the pressing need to address hospital wastewater as a crucial factor in curtailing the spread of carbapenem-resistant A. baumannii.

Analysis of resistance genes of carbapenem-resistant Providencia rettgeri using whole genome sequencing

ObjectiveThis study aimed to investigate the clinical infection characteristics and analyze the resistance gene carrying status of carbapenem-resistant Providencia rettgeri via whole genome sequencing (WGS).MethodsCarbapenem-resistant P. rettgeri were collected from clinical patients between January 2020 and December 2021, and their susceptibility to 19 antimicrobial drugs was determined using the VITEK 2 Compact system and Kirby–Bauer (KB) disk diffusion method. The Illumina platform was used to perform WGS of the P. rettgeri isolates, and the resistance genes carried by the Carbapenem-resistant P. rettgeri strains were detected via ABRicate software. The phylogenetic tree was constructed by thirty-four strains including twenty-eight strains downloaded from NCBI database and the carbapenem-resistant six P. rettgeri strains in this study. Which based on genomic single nucleotide polymorphism (SNP) to understand the affinities of the carbapenem-resistant P. rettgeri strains.ResultsSix carbapenem-resistant P. rettgeri strains were isolated from five different clinical departments using the blood, urine, sputum, and secretion specimens. These infected patients are middle-aged and elderly people with a history of severe trauma, tumors, hypertension, and various other underlying diseases, and invasive procedures. Antimicrobial sensitivity testing showed that all strains presented resistance to ampicillin-sulbactam, ceftazidime, ciprofloxacin, levofloxacin, and ertapenem, whereas they exhibited full susceptibility to cefepime and amikacin. Most strains demonstrated high resistance to β-lactams, aminoglycosides, and sulfonamides. Thirty-five resistance genes were identified by ABRicate. All carbapenem-resistant P. rettgeri strains carried aminoglycoside, fluoroquinolone, chloramphenicol, rifampicin, sulfonamide, and β-lactam resistance genes, and most importantly, all strains possessed the carbapenem resistance gene blaNDM−1. The six P. rettgeri strains in this study and the 28 carbapenem-resistant P. rettgeri strains from the NCBI database were divided into four evolutionary groups. The WF3643, WF3849, WF3822, and WF3821 strains in this study were in the same evolutionary group (clade A), while the closely related WF3099 and WF3279 strains were in different evolutionary groups (clade B and clade D), respectively. The WF3099 strain was distantly related to the other five strains.ConclusionCarbapenem-resistant P. rettgeri strains were mostly isolated from middle-aged and older patients with a history of surgery or serious underlying diseases, and they were found to cause multisystem infections. All Carbapenem-resistant P. rettgeri strains in this study carried blaNDM−1 and multiple antimicrobial drug resistance genes. Furthermore, the P. rettgeri strains in this study were closely related, suggesting the possibility of nosocomial infections. Therefore, our study highlights the need for research on P. rettgeri to control the spread of these nosocomial infections.

Whole-genome sequencing provides insights into a novel species: Providencia hangzhouensis associated with urinary tract infections.

Providencia rettgeri is a clinically significant opportunistic pathogen that is involved in urinary tract infections. Due to the resolution limitations of identification, distinguishing P. rettgeri from closely related species is challenging by commercial biochemical test systems. Here, we first reported a novel species, Providencia hangzhouensis, which had been misidentified as P. rettgeri. Exhibiting ≤91.97% average nucleotide identity (ANI) and ≤46.10% in silico DNA-DNA hybridization values with all known Providencia species, P. hangzhouensis falls well beneath the established species-defining thresholds. We conducted a population genomics analysis of P. hangzhouensis isolates worldwide. Our study revealed that P. hangzhouensis has emerged in many countries and has formed several transmission clusters. We found that P. hangzhouensis shared the highest ANI values (91.54% and 91.97%) with P. rettgeri and P. huaxiensis, respectively. The pan-genome analysis revealed that these three species possessed a similar component of pan-genomes. Two genes associated with metabolism, folE2 and ccmM, were identified to be specific to P. hangzhouensis. Furthermore, we also observed that carbapenem-resistance genes frequently occur in P. hangzhouensis with the blaIMP-27 being the most prevalent (46.15%; 36/78). The emergence of P. hangzhouensis is often accompanied by extended-spectrum β-lactamase and carbapenem-resistance genes, and calls for tailored surveillance of this species as a clinically relevant species in the future. IMPORTANCE Our study has identified and characterized a novel species, Providencia hangzhouensis, which is associated with urinary tract infections and was previously misidentified as Providencia rettgeri. Through this study, we have identified specific genes unique to P. hangzhouensis, which could serve as marker genes for rapid PCR identification. Additionally, our findings suggest that the emergence of P. hangzhouensis is often accompanied by extended-spectrum β-lactamase and carbapenem-resistance genes, emphasizing the need for attention to clinical management and the importance of accurate species identification and proper drug use.

Analysis of NDM-1 and IMP-8 carbapenemase producing Raoultella planticola clinical isolates.

Object of our study was to analyze the carriage of resistance genes in carbapenem-resistant Raoultella planticola (CRRP) by whole genome sequencing (WGS). Three strains of CRRP (named WF0027, WF3597 and WF3648) were collected for clinical analysis and susceptibility of antimicrobial agents was determined. The WGS of three strains was done by Illumina platform and strain identification was performed by average nucleotide identity, and the antibiotic resistance genes carried by the three strains were detected by ABRicate software. Whole genome data of 46 CRRP strains were downloaded from the National Center for Biotechnology Information (NCBI) database, and the evolutionary tree was constructed by genomic single nucleotide polymorphism together with this study strains. Antimicrobial susceptibility testing revealed that WF3597 and WF3648 were susceptible to tigecycline and colistin, while exhibited resistance to 24 antimicrobial agents. WF0027 was resistant to 18 antimicrobial agents. A total of 25 resistance genes were identified using ABRicate software. WF0027 carried blaIMP-8, whereas WF3597 and WF3648 carried blaNDM-1 carbapenem resistance gene. As predicted by the PlasmidFinder, WF3597 and WF3648 carried one plasmid IncFII(p14)_1_p14, whereas WF0027 carried five plasmids. Evolutionary tree results show all strains are clustered into six groups, the strains WF3597 and WF3648 belonged to the same evolutionary group (E clade) and WF0027 belonged to the F clade. Three CRRP strains in our study carried carbapenem resistance genes (blaNDM-1 or blaIMP-8) and were resistant to multiple antimicrobial agents, posing a significant challenge for clinical treatment.

Performance of Carbapenemase Nordmann-Poirel, Modified Carbapenem Inactivation, and EDTA Carbapenem Inactivation Methods for Detecting Carbapenem-Resistant Klebsiella pneumoniae Isolates.

Infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP) are a major threat to public health. Timely detection of CRKP will help treat patients with appropriate antibiotics. This study aimed to evaluate the performance of the carbapenemase Nordmann-Poirel (CarbaNP), modified carbapenem inactivation (mCIM), and EDTA carbapenem inactivation (eCIM) methods for the detection of CRKP. We compared the results of the three assays with that of real-time PCR. In total, 195 K. pneumoniae isolates, including 150 carbapenem-resistant and 45 carbapenem-susceptible isolates, were investigated. Carbapenem-resistance genes, such as blaKPC, blaNDM, blaVIM, blaIMP, and blaOXA-48-like, were identified using real-time PCR. Among the 150 CRKP isolates, 94 (62.7%) were positive for blaNDM, 29 (19.3%) were positive for blaOXA-48-like, and 27 (18%) were positive for both blaNDM and blaOXA-48-like. For detecting CRKP isolates, CarbaNP, mCIM, and eCIM showed 96.0%, 95.4%, and 96.7% sensitivity, respectively, and all three methods showed 100% specificity. All three phenotypic confirmatory tests are reliable for identifying CRKP, easy to perform, cost-effective, and can be incorporated with routine antibiotic susceptibility testing.

Emergence of a clinical isolate of E. coil ST297 co-carrying blaNDM-13 and mcr-1.1 in China

BackgroundThe coexistence of carbapenem resistance genes and mcr-1.1 in Enterobacterales has been an urgent and persistent threat to global public health. In this study, we isolated a clinical NB4833, an Escherichia coli isolate that co-carries mcr-1.1 and blaNDM-13. ObjectivesThis study aimed to isolate a clinical NB4833, an Escherichia coli isolate that co-carries mcr-1.1 and blaNDM-13 and investigated the phenotypic and genotypic characteristics of plasmids harbored by E. coli isolate NB4833. MethodsAntimicrobial susceptibility testing and conjugation assay were performed on E. coli isolate NB4833. Stability of the plasmid and growth rate determination were used to characterize the plasmids harboring mcr-1.1 and blaNDM-13. In addition, the genetic characteristics of the plasmids were analyzed based on whole-genome sequencing of the strain and comparative genetic analysis with related plasmids. ResultsWhole-genome sequencing showed that the isolate carried multiple resistance genes and possessed phenotypes indicative of all antibiotic resistance except tigecycline. And the mcr-1.1- and blaNDM-13-harbouring plasmids showed relatively high similarity to the related plasmids. The pNB4833-NDM-13 plasmid was capable of trans conjugation with an efficiency of 1.04 × 10−2 in a filter mating experiment and the transconjugant J53/ pNB4833-NDM-13 was able to be stably inherited after 10 days of passage. ConclusionsTo our knowledge, this is the first report of the coexistence of the IncI2 plasmid carrying mcr-1.1 and a blaNDM-13-carrying integrated IncFIB/IncFII plasmid in an ST297 clinical E. coli isolate. In addition, we investigated a novel plasmid carrying blaNDM-13. Our study expands the diversity of plasmids carrying blaNDM-13, which exhibits epidemic importance in bacterial resistance. Therefore, there are important measures that should be taken to prevent the spread of these plasmids.

Characterisation of a Novel Tigecycline Resistance Gene tet(X22) and its Coexistence with blaNDM-1 in a Pseudomonas caeni Isolate

ObjectivesThe emergence of pathogens that are resistant to both tigecycline and carbapenem poses a threat to public health globally. Continuous emergence of novel tet(X) variants accelerates the tigecycline resistance crisis. This study aimed to characterise the novel tigecycline resistance gene tet(X22) and its coexistence with carbapenem resistance gene blaNDM-1 in Pseudomonas caeni. MethodsThis P. caeni isolate co-harbouring tet(X22) and blaNDM-1 was systematically investigated using antimicrobial susceptibility testing, conjugation assays, genome sequencing, bioinformatic analyses, cloning of tet(X22) and functional analysis, and protein structure prediction. ResultsThe carbapenem-resistant and tigecycline-resistant P. caeni isolate CE14 was obtained from chicken faeces in 2022. CE14 carried multiple antibiotic resistance genes, including the novel tet(X22) and blaNDM-1. Tet(X22) exhibited 64.72–90.48% amino acid identity with other variants [Tet(X) to Tet(X21)]. Cloning of the gene tet(X22) and protein structure prediction revealed that Tet(X22) confers resistance to tetracyclines, including tigecycline. tet(X22) and blaNDM-1 were located in two multidrug-resistant regions of the chromosome. ConclusionsThe occurrence of the novel ISCR2-flanked tet(X22) in P. caeni suggests that the tet(X) variant has adapted to new hosts and may widely spread to further expand the host range. The future global spread of such pathogens co-harbouring tet(X) and blaNDM variants needs to be continuously monitored according to the One Health approach.

Identification of TMexCD-TOprJ-producing carbapenem-resistant Gram-negative bacteria from hospital sewage.

Carbapenems and tigecycline are crucial antimicrobials for the treatment of gram-negative bacteria infections. Recently, a novel resistance-nodulation-division (RND) efflux pump gene cluster, tmexCD-toprJ, which confers resistance to tigecycline, has been discovered in animals and clinical isolates. It was reported that hospital sewage could act as a reservoir for gram-negative bacteria with high antimicrobial resistance genes. In this study, we analyzed 84 isolates of carbapenem-resistant gram-negative bacteria (CR-GNB) from hospital sewage, and identified five isolates of TMexCD-ToprJ-producing CR-GNB, including one Raoultella ornithinolytica isolate and four Pseudomonas spp. isolates. All these five isolates carried at least one carbapenem resistance gene and were resistant to multiple antibiotics. Multiple tmexCD-toprJ clusters were detected, including tmexC2D2-toprJ2, tmexC3D3-toprJ3, tmexC3.2D3.3-toprJ1b and tmexC3.2D3-toprJ1b. Among these clusters, the genetic construct of tmexC3.2D3-toprJ1b showed 2-fold higher minimum inhibitory concentration (MIC) of tigecycline than other three variants. In addition, it was found that the tmexCD-toprJ gene cluster was originated from Pseudomonas spp. and mainly located on Tn6855 variants inserted in the same umuC-like genes on chromosomes and plasmids. This unit co-localized with blaIMP or blaVIM on IncHI5-, IncpJBCL41- and IncpSTY-type plasmids in the five isolates of TMCR-GNB. The IncHI5- and IncpSTY-type plasmids had the ability to conjugal transfer to E. coli J53 and P. aeruginosa PAO1, highlighting the potential risk of transfer of tmexCD-toprJ from Pseudomonas spp. to Enterobacterales. Importantly, genomic analysis showed that similar tmexCD-toprJ-harboring IncHI5 plasmids were also detected in human samples, suggesting transmission between environmental and human sectors. The emergence of TMCR-GNB from hospital sewage underscores the need for ongoing surveillance of antimicrobial resistance genes, particularly the novel resistance genes such as the tmexCD-toprJ gene clusters in the wastewater environment.

Do Pathogenic Escherichia coli Isolated from Gallus gallus in South Africa Carry Co-Resistance Toward Colistin and Carbapenem Antimicrobials?

Colistin and carbapenems are critically important antimicrobials often used as a last resort to manage multidrug-resistant bacterial infections in humans. With limited alternatives, resistance to these antimicrobials is of concern as organisms could potentially spread horizontally rendering treatments ineffective. The aim of this study was to investigate co-resistance to colistin and carbapenems among Escherichia coli isolated from poultry in South Africa. Forty-six E. coli strains obtained from clinical cases of breeder and broiler chickens were used. In addition to other antibiotics, all the isolates were tested against colistin and carbapenems using broth microdilution. Multiplex polymerase chain reactions were used to investigate the presence of colistin (mcr-1 to 5) and carbapenem (blaOXA-48, blaNDM-1, and blaVIM) resistance genes. Isolates exhibiting colistin resistance (>2 μg/mL) underwent a whole-genome sequencing analysis. Resistance to colistin (10.9%) and cefepime (6.5%) was noted with all colistin-resistant strains harboring the mcr-1 gene. None of the E. coli isolates were resistant to carbapenems nor carried the other resistant genes (mcr-2 to 5, blaOXA-48, blaNDM-1, and blaVIM). The mcr-1-positive strains belonged to sequence types ST117 and ST156 and carried virulence genes ompA, aslA, fdeC, fimH, iroN, iutA, tsh, pic, ast A and set 1A/1B. In conclusion, clinical E. coli strains from chickens in this study possessed mobile resistance genes for colistin and several other clinically relevant antimicrobials but not carbapenems. Additionally, they belonged to sequence types in addition to carrying virulence factors often associated with human extraintestinal pathogenic E. coli infections. Thus, the potential risk of transmitting these strains to humans cannot be underestimated especially if sick birds are dispatched into the thriving poorly regulated Cornish hen industry. The need for routine veterinary surveillance and monitoring of antimicrobial resistance, antimicrobial use and the importance of strengthening regulations guiding the informal poultry sector remains important.

Genomic Analysis of Carbapenem-Resistant Hypervirulent Klebsiella pneumoniae in a Chinese Tertiary Hospital.

Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) has become a clinical crisis and is associated with significant morbidity and mortality. The prevalence of CR-hvKP has trended upward since 2010. This study aims to describe the clinical and genomic characteristics of CR-hvKP collected from a tertiary hospital in eastern China, from August 2020 to October 2021. We tested the susceptibility to common antibiotics in these isolates to feature the antibiotic-resistant phenotypes. We also applied whole-genome sequencing and core-genome phylogenetic to analysis the genetic features of these isolates. Plasmid replicons were identified by using the PlasmidFinder database, and core-genome phylogenetic analysis by Parsnp database. All these strains isolated from the patients with serious underlying diseases and poor prognosis. We found all CR-hvKp isolates exhibited a multidrug-resistant (MDR) phenotype. These results revealed that blaKPC-2 was the predominant carbapenemases gene (n = 53, 84.1%), and ST11-KL64 CR-hvKP strains dominated, forming a single cluster, and differed by an average of 26 core SNPs. We only found eight ST15 isolates containing KL24 and KL112 type capsules, with the main carbapenem resistance genes being blaOXA-232 and blaKPC-2. All ST11-KL64 strains had a series of resistance and virulence genes, along with IncHIB-FIB virulence plasmids and IncFII resistance plasmids, while the prevalence of resistance plasmids like the IncFII plasmid was absence in ST15 isolates. This suggests that ST11-KL64 CR-hvKP has emerged as the most prevalent hypervirulence and carbapenem-resistant K. pneumoniae and may contribute to hospital outbreaks of infection, which required most clinical attention.

Carbapenem Resistance Mechanisms in Acinetobacter baumannii: Spotlight on OXA Genes

Acinetobacter baumannii is a highly problematic bacterium due to its multidrug-resistant nature and ability to rapidly develop resistance. Carbapenem-resistant A. baumannii (CRAB) is particularly concerning, recognized as a top priority pathogen by the World Health Organization (WHO). In this study, we aimed to assess the susceptibility of A. baumannii to antibiotics and characterize the presence of specific carbapenem resistance genes using PCR analysis. The study was conducted as a cross-sectional investigation at Al-Sader Medical City and Baghdad Teaching Hospitals between October 2022 and February 2023. A total of 59 A.baumannii isolates were collected from patients. To ensure accuracy in diagnosis, the isolates underwent thorough processing using morphological techniques, biochemical tests, and Vitek2 systems. The susceptibility of the isolates to 24 antibiotics was evaluated using the Kirby-Bauer method. Additionally, DNA extraction and PCR analysis were performed to detect the presence of carbapenem resistance genes. The study included analysis of 59 specimens collected from patients, including sputum, wound swabs, blood, and inguinal swabs. The majority of the isolates were obtained from in-patients, demonstrating a significant difference compared to outpatients. Among the studied isolates, the most prevalent carbapenem resistance gene detected was blaOXA51, present in 97% of the isolates. This highlights the substantial presence of blaOXA-mediated carbapenem resistance among A. baumannii strains in our study population. Furthermore, our findings revealed alarmingly high resistance rates against the majority of antibiotics commonly used to treat A. baumannii infections. Approximately 95% of the isolates were classified as extensively drug-resistant, indicating resistance to multiple classes of antibiotics. This poses significant challenges in terms of effective treatment options and emphasizes the urgent need for alternative strategies to manage A. baumannii infections.

Isolation and Characterisation of Human-Derived blaKPC-3-Producing Salmonella enterica Serovar Rissen in 2018.

In this study, we describe a Salmonella enterica serovar (S.) Rissen strain with a reduced susceptibility to meropenem, isolated from a urinary infection in an 89-year-old woman in 2018 during activity surveillance in Italy (Enter-Net Italia). The genomic characteristics, pathogenicity, and antimicrobial resistance mechanisms were investigated via a genomic approach. Antimicrobial susceptibility testing revealed a "susceptible, increased exposure" phenotype to meropenem in the S. Rissen strain (4_29_19). Whole-genome sequencing (WGS) was performed using both the NovaSeq 6000 S4 PE150 XP platform (Illumina, San Diego, CA, USA) and MinION (Oxford Nanopore). The S. Rissen 4_29_19 strain harboured two plasmids: a pKpQIL-like plasmid carrying the blaKPC-3 resistance gene in a Tn4401a transposon (pKPC_4_29_19), and a ColE-like plasmid (p4_4_29_19) without resistance genes, highly prevalent among Enterobacterales. Comparative analysis revealed that the pKPC_4_29_19 plasmid was highly related to the pKpQIL reference plasmid (GU595196), with 57% coverage and 99.96% identity, but lacking a region of about 30 kb, involving the FIIK2 replicon region and the entire transfer locus, causing the loss of its ability to conjugate. To our knowledge, this is the first time that a pKpQIL-like plasmid, carrying blaKPC-3, highly diffused in Klebsiella pneumoniae strains, has been identified in a Salmonella strain in our country. The acquisition of blaKPC genes by Salmonella spp. is extremely rare, and is reported only sporadically. In zoonotic bacteria isolated from humans, the presence of a carbapenem resistance gene carried by mobile genetic elements, usually described in healthcare-associated infection bacteria, represents an important concern for public health.

Molecular characterization and comparison of bla NDM-1-carrying and bla NDM-5-harboring IncX3-type plasmids in carbapenem-resistant Klebsiella pneumoniae.

Carbapenem-resistant Klebsiella pneumoniae (CRKP), which harbors the bla NDM plasmid, has been reported extensively and is considered a global threat clinically. However, characterization and comparisons of bla NDM-1-carrying and bla NDM-5-harboring IncX3-type plasmids in CRKP are lacking. Here, we systematically compared the differences in the characteristics, genetic backgrounds, transferability, and fitness costs between bla NDM-1-carrying and bla NDM-5-carrying plasmids in K. pneumoniae isolates. Fifteen NDM-producing CRKP isolates were recovered from 1376 CRKP isolates between 2019 and 2021, of which 4 were positive for bla NDM-1 and 11 were positive for bla NDM-5. All strains were highly resistant to carbapenem but remained susceptible to tigecycline and colistin. Core-genome-based phylogenetic analyses revealed that these strains were not clonally related. Whole-genome sequencing showed that bla NDM-1 and bla NDM-5 were located on ~54 kb and ~46 kb IncX3-type plasmids, respectively. The backbone, genetic context, and fitness cost of the bla NDM-1-bearing plasmid were highly similar to those of the bla NDM-5-carrying plasmid, but the transferability of the bla NDM-1-positive plasmid was greater than that of the bla NDM-5-positive plasmid. In conclusion, the transmission of bla NDM-1 or bla NDM-5 is mainly disseminated by plasmids rather than clonal spread. The high transfer frequency of the IncX3 plasmid facilitates the prevalence and dissemination of NDM-KP among Enterobacteriaceae. IMPORTANCE The emergence of NDM-producing Klebsiella pneumoniae is a severe challenge to public health. The widespread presence of bla NDM-1 and bla NDM-5 in Enterobacteriaceae has aroused broad concern. In this study, we performed molecular characterization of bla NDM-1-carrying and bla NDM-5-harboring IncX3-type plasmids in carbapenem-resistant Klebsiella pneumoniae (CRKP) and compared their phenotypes between strains with different bla NDM subtype. Our findings highlight the importance of IncX3-type plasmids in the transfer of the bla NDM-1 and bla NDM-5 genes and demonstrate that the bla NDM-1 plasmid possesses higher transfer ability. These data will provide important insights into carbapenem resistance gene transfer via plasmids and their further spread in clinical settings.

Transmission of clones of carbapenem-resistant Escherichia coli between a hospital and an urban wastewater treatment plant

Carbapenem-resistant Enterobacterales (CRE) constitute an urgent threat to worldwide public health. The spread of CRE is facilitated by transmission via the environment. Wastewater treatment plants (WWTPs) are considered to be important sources of antibiotic resistance and hot spots of antibiotic-resistant bacteria (ARB) which can facilitate dissemination of antibiotic resistance genes. In this study, water samples were collected over one year from a WWTP in Jinan, Shandong province, China, from different functional sites in the wastewater treatment process. Carbapenem-resistant Escherichia coli (CREC) were isolated by selective cultivation and whole-genome sequenced to investigate the occurrence and characteristics of CREC in the WWTP. A total of 77 CREC isolates were included in the study and the detection rate of CREC in the WWTP water inlet was found to be 85%. An additional 10 CREC were isolated from a nearby teaching hospital during the sampling period and included for comparison to the environmental isolates. Susceptibility testing showed that all CREC were multidrug-resistant. 6 different carbapenem resistance genes (CRGs) were detected, including blaNDM-5 (n = 75), blaNDM-1 (n = 6), blaNDM-4 (n = 3), blaNDM-6 (n = 1), blaNDM-9 (n = 1), and blaKPC-2 (n = 4). 42 CREC isolates were whole-genome sequenced with Illumina short-read sequencing. 11 of these were also sequenced with Nanopore long-read sequencing. Plasmids carrying CRGs were found to belong to IncX3 (n = 35), IncFII (n = 12), IncFIA (n = 5), IncFIB (n = 2), IncC (n = 1), and IncP6 (n = 1). Clonal dissemination of CREC belonging to ST167, ST448, and ST746 was observed between different parts of the WWTP. Furthermore, isolates from the WWTP, including an isolate belonging to the high-risk ST167 strain, were found to be clonally related to CREC isolated at the hospital. The spread of CRGs is of considerable concern and strategies to prevent environmental dissemination of this contaminant urgently needs to be implemented.