Conferring Carbapenem Resistance Research Articles

Risk Factors and Outcomes of Patients Colonized with KPC and NDM Carbapenemase-Producing Enterobacterales.

Carbapenemase-producing enterobacterales (CPE) poses an increasing threat in hospitals worldwide. Recently, the prevalence of different carbapenemases conferring carbapenem resistance in enterobacterales changed in our country, including an increase in New Delhi Metallo-beta-lactamase (NDM)-CPE. We conducted a comparative historical study of adult patients colonized with Klebsiella pneumoniae carbapenemase (KPC)-CPE (July 2016 to June 2018, a historical cohort) vs. NDM-CPE (July 2016 to January 2023). We identified patients retrospectively through the microbiology laboratory and reviewed their files, extracting demographics, underlying diseases, Charlson Comorbidity Index (CCI) scores, treatments, and outcomes. This study included 228 consecutive patients from whom a CPE rectal swab screening was obtained: 136 NDM-CPE positive and 92 KPC-CPE positive. NDM-CPE-colonized patients had a shorter hospitalization length and a significantly lower 30-day post-discharge mortality rate (p = 0.002) than KPC-CPE-colonized patients. Based on multivariate regression, independent risk factors predicting CPE-NDM colonization included admission from home and CCI < 4 (p < 0.001, p = 0.037, respectively). The increase in NDM-CPE prevalence necessitates a modified CPE screening strategy upon hospital admission tailored to the changing local CPE epidemiology. In our region, the screening of younger patients residing at home with fewer comorbidities should be considered, regardless of a prior community healthcare contact or hospital admission.

Molecular characteristics of carbapenem-resistant Enterobacteriaceae isolated from two tertiary hospitals in the Philippines

Species belonging to the family Enterobacteriaceae cause infections that result in clinically significant morbidity and mortality. These organisms have been shown to harbor multiple mechanisms of resistance to antibiotics, including carbapenems. Molecular characterization of resistance to carbapenems is critical since few effective therapeutic options exist, most of which are difficult to access in resource-limited settings. One hundred twenty-six Enterobacteriaceae isolates with phenotypic resistance to at least one carbapenem were collected from two tertiary hospitals in the Philippines from 2014 to 2016. The organisms were screened for the presence of genes coding for carbapenemases and extended spectrum β-lactamases (ESBLs) using multiplex PCR. Multilocus sequence typing (MLST) was performed for Klebsiella pneumoniae, Escherichia coli and Enterobacter cloacae complex. The majority of the carbapenem-resistant isolates were K. pneumoniae (70/126, 55.5%) followed by K. ozaenae (19/126, 15%). Amplification of genes conferring carbapenem resistance showed blaNDM in 89 isolates (70%), followed by blaKPC in 21 isolates (16%). MLST revealed the presence of different clones including ST16 (5/70), ST17 (3/70) and ST147 (9/70) in K. pneumoniae, and ST10 (3/13) in E. coli. This study shows that blaNDM is the most prevalent carbapenemase type among carbapenem-resistant Enterobacteriaceae (CRE). Presence of carbapenemase gene and multiple ESBLs in the same organism pose a risk in the clinical setting since it could confer higher antibiotic resistance. This high rate of resistance poses the risk of spread since it can be transferred by mobile genetic elements. This study illustrates the acute need for effective therapeutic and hospital infection controls against CREs.

Characterization of an ST38 carbapenem-resistant and highly virulent Escherichia coli carrying conjugatively transferable ColV virulence-resistance and blaNDM-5-positive resistance plasmids.

To characterize an Escherichia coli strain causing bloodstream infection encoding both high-virulence and carbapenem-resistance phenotypes. Antimicrobial susceptibility testing, WGS and bioinformatics analysis were performed to characterize strain E1. The function of the ColV plasmid was investigated by the Galleria mellonella infection model, serum killing and macrophage killing assays. The fitness effect of the ColV plasmid was tested by growth curve, plasmid stability tests and the in vitro competition assay. The conjugation assay was performed to test the transferability of the ColV and blaNDM-5-carrying plasmids. E. coli E1 from bloodstream infection was MDR and highly virulent in the G. mellonella infection model. It belonged to phylogroup D, ST38 and serotype O7:H8. E1 carried a conjugatively transferable IncI1-type blaNDM-5-positive plasmid, which conferred carbapenem resistance, a conjugative IncFIB/FII-type ColV plasmid encoding an array of virulence-associated genes and antibiotic resistance genes blaTEM-1B, strAB and sul2, and seven other plasmids. Co-transfer of the ColV plasmid and the blaNDM-5-positive plasmid was observed. The ColV virulence-resistance hybrid plasmid contributed to the virulence, resistance to serum killing, and macrophage phagocytosis in E. coli E1. The carriage of this ColV plasmid did not constitute an in vitro fitness burden to strain E1 but caused fitness costs to E. coli strain EC600. The emergence of such a highly virulent and resistant strain with conjugative blaNDM-5-positive and ColV plasmids posed a significant threat to public health. Implementation of control measures is needed to prevent such strains from further disseminating in hospital settings and the community.

The dynamic evolution and IS26-mediated interspecies transfer of a blaNDM-1-bearing fusion plasmid leading to a hypervirulent carbapenem-resistant Klebsiella pneumoniae strain harbouring blaKPC-2 in a single patient

To characterize the evolution and interspecies transfer of plasmids between Klebsiella pneumoniae and Escherichia coli within a single patient. Minimum inhibitory concentrations were measured using broth microdilution assays. Conjugation assays, string tests, and Galleria mellonella infection model experiments were also conducted. Whole-genome sequencing was performed on the Illumina and Nanopore platforms. Antimicrobial resistance determinants, insertion sequences, and virulence factors were identified using ABRicate/ResFinder database, ISFinder, and virulence factor database. Wzi and capsular polysaccharide (KL) were typed using Kleborate and Kaptive. Multi-locus sequence typing (MLST), replicon typing, and single nucleotide polymorphism analyses were conducted using the BacWGSTdb server. The carbapenem-resistant K. pneumoniae 2111KP was characterized as ST11, wzi64, and KL64, with a positive string test result and a relatively high virulence phenotype. Analysis of the 2111KP genome revealed that blaNDM-1 was located in a 268,400-bp IncFIB/IncHI1B/IncX3 conjugative plasmid (p2111KP-1), regulated by IS26, IS5, and ISKox3. p2111KP-1 was also a rmpA2-associated virulence plasmid with an iutA-iucABCD gene cluster and a IS26-mediated multidrug-resistant fusion plasmid, which contained 8-bp (AGCTGCAC or GGCCTTTG) target site duplications. Segments flanked by IS26 of p2111KP-1 were 99.99% identical to a 49,016-bp E. coli plasmid. This study provided direct evidence of plasmid fusion via IS26 between two different bacterial species within one patient and revealed the process by which genetic elements conferring carbapenem resistance and virulence were simultaneously transferred between these species. It highlights the need for strategic antibiotic use and rigorous monitoring to prevent the plasmid-mediated fusion and transmission of drug-resistance/virulence factors.

Anthropogenic and natural disturbances along a river and its estuary alter the diversity of pathogens and antibiotic resistance mechanisms

Antibiotic resistance (AMR) in pathogens threatens human health worldwide, and antibiotic-resistant bacteria (ARB) are widespread in the environment. In particular, anthropogenically-disturbed rivers became reservoirs of ARBs and hotspots of antibiotic resistance gene (ARG) transmission. However, the diversity and sources of ARB, and the mechanisms of ARG transmission are not fully known. Here, we used deep metagenomic sequencing to study the dynamics of pathogens and their antibiotic resistance mechanisms along the Alexander River (Israel), affected by sewage and animal farm runoffs. Putative pathogens such as Aeromicrobium marinum and Mycobacterium massilipolynesiensis were enriched in western stations, following the inputs of polluted Nablus River. Aeromonas veronii was dominant in eastern stations in Spring. Several AMR mechanisms showed distinct patterns in Summer-Spring (dry season) and Winter (rainy season). We found low abundance beta-lactamases conferring carbapenem resistance: e.g., OXA-912 was linked to A. veronii in Spring; OXA-119 and OXA-205 to Xanthomonadaceae in Winter. We classified 33 % of ARG-containing contigs as putative plasmid sequences, indicating the high potential for resistome transmission. A limited number of ARGs were linked to putative phages. Our results suggest that this model river is a hotspot for AMR activity and transmission, and highlight the merit of deep sequencing for AMR discovery.

Development of an immunochromatographic lateral flow assay to rapidly detect OXA-23-, OXA-40-, OXA-58- and NDM-mediated carbapenem resistance determinants in Acinetobacter baumannii.

Introduction. Acinetobacter baumannii infections can be extremely challenging to treat owing to the worldwide prevalence of multidrug-resistant isolates, especially against carbapenems. Colonization with carbapenem-resistant A. baumannii (CRAb) requires rapid action from an infection control perspective because the organism is known for its propensity for epidemic spread. Hypothesis/Gap Statement. There is an unmet medical need to rapidly identify CRAb to enable appropriate antimicrobial treatment and to prevent transmission. Aim. Our aim was to expand the OXA-detection abilities of the rapid immunochromatographic test (ICT) OXA-23 K-SeT (Coris BioConcept) to include OXA-40- and OXA-58-like carbapenemases, which together confer carbapenem resistance to more than 94 % of CRAb isolates worldwide. Methodology. We used hybridoma technology to generate mAbs against OXA-40 and OXA-58 and selected them for productivity and specificity against recombinant and endogenous OXA-40 and OXA-58. Combinations of the resulting mAbs were analysed in ICT format for their ability to detect recombinant rOXA-40His6 or rOXA-58His6, respectively. Subsequently, selected antibody pairs were implemented into single-OXA-40 or single-OXA-58 prototypes and the final OXA-23/40/58/NDM ICT and were evaluated on clinical Acinetobacter spp. isolates with well-defined carbapenem resistance mechanisms. Results. Five anti-OXA-40 and anti-OXA-58 mAbs were selected. Competition ELISA with combinations of these antibodies revealed that the anti-OXA-40 antibodies bind to one of two binding clusters on OXA-40, while anti-OXA-58 antibodies bind to one of four binding clusters on OXA-58. Direct binding to the corresponding antigen in an ICT format has left only three antibodies against rOXA-40His6 and rOXA-58His6, respectively for the subsequent sandwich ICT selection procedure, which revealed that the anti-OXA-40 (#5) and anti-OXA-58 (#A8) mAbs in combination with the cross-reactive mAb #C8 performed best. They were implemented into single-OXA-40 and single-OXA-58 ICT prototypes and evaluated. These single ICT prototypes demonstrated 100 % specificity and sensitivity. Based on these results, an OXA-23/40/58/NDM-ICT was developed, complemented with OXA-23 and NDM-specific detection. An evaluation with selected carbapenem-resistant Acinetobacter spp. isolates (n=34) showed 100 % specificity. Conclusion. With this easy-to-use detection assay, one can save 12-48 h in diagnostics, which helps to treat patients earlier with appropriate antibiotics and allows immediate intervention to control transmission of CRAb.

Presence of Polyketide Synthase (PKS) Gene and Counterpart Virulence Determinants in Klebsiella pneumoniae Strains Enhances Colorectal Cancer Progression In-Vitro.

Klebsiella pneumoniae (K. pneumoniae) colonizes the human gut and is a causative factor of pyogenic liver abscess (PLA). Retrospective studies conducted on K. pneumoniae PLA patients revealed subsequent CRC development in later years of their life with increasing prevalence of these strains harbouring polyketide synthase (PKS) genes. To our knowledge there are no known studies directly implicating K. pneumoniae with CRC to date. Our aims are to characterize K. pneumoniae isolates from CRC patients and investigate its effects on cell proliferation in vitro. K. pneumoniae isolates were characterized by screening virulence genes including polyketide synthase (PKS), biofilm assay, antibiotic susceptibility, and string test to determine hypervirulent (hvKp) strains. Solubilised antigens of selected K. pneumoniae isolates were co-cultured with primary colon cell lines and CRC cell lines (Stage I-IV) for 48 h. The enhancement of proliferation was measured through MTT and ECIS assay. Twenty-five percent of K. pneumoniae isolates were PKS-positive out of which 50% were hvKp strains. The majority of the isolates were from the more virulent serotype of K1 (30%) and K2 (50%). PKS-positive K. pneumoniae isolates did not possess genes to confer carbapenem resistance but instead were more highly associated with siderophore genes (aerobactin, enterobactin, and yersiniabactin) and allantoin metabolism genes (allS, allS2). Cell proliferation in primary colon, SW1116 (Stage I), and SW480 (Stage II) CRC cell lines were enhanced when co-cultured with PKS-positive K. pneumoniae antigens. ECIS revealed enhanced cell proliferation upon recurrent antigen exposure. This demonstrates the possible role that PKS-positive K. pneumoniae has in exacerbating CRC progression.

Characterisation of Non-Carbapenemase-Producing Carbapenem-Resistant Klebsiella pneumoniae Based on Their Clinical and Molecular Profile in Malaysia.

Non-carbapenemase-producing carbapenem-resistant Klebsiella pneumoniae (NC-CRKP) confers carbapenem resistance through a combination of chromosomal mutations and acquired non-carbapenemase resistance mechanisms. In this study, we aimed to evaluate the clinical and molecular profiles of NC-CRKP isolated from patients in a tertiary teaching hospital in Malaysia from January 2013 to October 2019. During the study period, 54 NC-CRKP-infected/colonised patients' isolates were obtained. Clinical parameters were assessed in 52 patients. The all-cause in-hospital mortality rate among NC-CRKP patients was 46.2% (24/52). Twenty-three (44.2%) patients were infected, while others were colonised. Based on the Charlson Comorbidity Index (CCI) score, 92.3% (48/52) of the infected/colonised patients had a score of ≥ 1. Resistance genes found among the 54 NC-CRKP isolates were blaTEM, blaSHV, blaCTX-M, blaOXA, and blaDHA. Porin loss was detected in 25/54 (46.3%) strains. None of the isolated strains conferred carbapenem resistance through the efflux pumps system. In conclusion, only 25/54 (46.3%) NC-CRKP conferred carbapenem resistance through a combination of porin loss and the acquisition of non-carbapenemase resistance mechanisms. The carbapenem resistance mechanisms for the remaining strains (53.7%) should be further investigated as rapid identification and distinction of the NC-CRKP mechanisms enable optimal treatment and infection control efforts.

Community-Acquired Acinetobacter radioresistens Bacteremia in an Immunocompetent Host

Acinetobacter species are gram-negative coccobacilli ubiquitousin nature and widely distributed in the environment. Acinetobacter baumannii isa bacteria commonly seen in the hospital setting, responsible for causinga wide range of bloodstream infections, urinary tract infections, secondary meningitis, infective endocarditis, and wound infections, and is the cause of outbreaks mainly due to its antimicrobial resistance patterns. The use of broad-spectrum antibiotic coverage with carbapenems is essential in the hospital setting. Therefore, carbapenem-resistantAcinetobacter baumannii(CRAB) poses as a very challenging pathogen.Acinetobacter radioresistens, a rare species in comparison to the more prevalentAcinetobacter baumannii, isan underestimated agent in causing nosocomial infections and also is a potential disseminator of resistance genes.It is also resistant to gamma radiation at 4-8 times higher than other Acinetobacter spp. andis the source of the class D OXA-23 carbapenemase that can confer carbapenem resistance. Therefore,immediate and precise identification of A. radioresistens is crucial for the clinical management of multidrug-resistant bacteremia.

A clinical Pseudomonas juntendi strain with bla IMP-1 carried by an integrative and conjugative element in China.

ObjectiveTo precisely determine the species of a carbapenem-resistant Pseudomonas strain 1809276 isolated from the urine of a Chinese patient and analyze its integrative and conjugative element (ICE) 1276 formation mechanism.MethodsSingle-molecule real-time (SMRT) sequencing was carried out on strain 18091276 to obtain the complete chromosome and plasmid (pCN1276) sequences, and average nucleotide identity (ANI) was used for precise species identification. The ICEs in GenBank with the same integrase structure as ICE 1276 were aligned. At the same time, the transfer ability of blaIMP−1 and the antibiotic sensitivity of Pseudomonas juntendi 18091276 were tested.ResultsThis bacterium was P. juntendi, and its drug resistance mechanism is the capture of the accA4' gene cassette by the Tn402-like type 1 integron (IntI1-blaIMP−1) to form In1886 before its capture by the ΔTn4662a-carrying ICE 1276. The acquisition of blaIMP−1 confers carbapenem resistance to P. juntendi 18091276.ConclusionThe formation of blaIMP−1-carrying ICE 1276, its further integration into the chromosomes, and transposition and recombination of other elements promote bacterial gene accumulation and transmission.

A novel Bacteroides metallo-β-lactamase (MBL) and its gene (crxA) in Bacteroides xylanisolvens revealed by genomic sequencing and functional analysis.

ObjectivesWe sought to characterize the carbapenem resistance mechanism of Bacteroides xylanisolvens 14880, an imipenem-resistant strain from Germany, and assess its prevalence.MethodsAntimicrobial susceptibilities were determined using agar dilution or Etest methodology and specific imipenemase activity was detected. The genomic sequence of B. xylanisolvens 14880 was determined and analysed for antibiotic resistance genes and genomic islands. We also used gene transfer to a carbapenem susceptible host, along with 5′-RACE, conventional PCR with capillary sequencing and RT–PCR-based screening.Results B. xylanisolvens 14880 displayed resistance to carbapenems and produced high specific imipenemase activity. Its genomic sequence was 6.1 Mbp and a class B1 β-lactamase gene (termed crxA) was detected in it. crxA was carried on a putative genomic island with insertion sequence (IS) elements and a putative GNAT (Gcn5-like acetyltransferase) toxin gene. Promoter localization by 5′-RACE and gene targeting to an imipenem-susceptible Bacteroides host indicated that it is activated by an IS1380-like IS element and it can confer carbapenem resistance. The PCR screening of Bacteroides strains showed that crxA was specific to B. xylanisolvens with a carriage rate of 16.7%.Conclusions B. xylanisolvens strains can harbour a carbapenem resistance gene, which has many similarities to the ‘cfiA system’: metallo-β-lactamase (MBL), IS element activation, carriage of a GNAT toxin gene, specific for a unique Bacteroides species with a significant prevalence.

Epidemiology and resistance mechanisms of tigecycline- and carbapenem-resistant Enterobacter cloacae in Southwest China: a 5-year retrospective study

The prevalence and molecular epidemiology of tigecycline resistance in carbapenem-resistant Enterobacter cloacae (CREC) in mainland China is unknown. We aimed to investigate the molecular characteristics and mechanisms of tigecycline-resistant CREC (TCREC) in Southwest China. We conducted a 5-year retrospective study. TCREC isolates underwent antimicrobial susceptibility testing, PFGE and MLST. We determined the presence of genes, deficiency of outer membrane proteins (OMPs) and expression of efflux pumps using PCR, reverse transcription PCR and SDS-PAGE. A large proportion of CREC isolates (21.7%; 36/166) were TCREC. All isolates were resistant to ertapenem, whereas 67% remained susceptible to imipenem and meropenem. ST88 (10/36; 27.8%) was predominant and was associated with moderate resistance to tigecycline and high resistance to carbapenems, followed by ST256 (3/36; 8.3%), ST78 (2/36; 5.6%), ST557 (2/36; 5.6%) and ST102 (2/36; 5.6%). blaNDM-1 (6/36; 16.7%) was the most common carbapenemase gene, and ST88 (5/6; 83.3%) was the most common type, followed by blaIMP-8 (3/36; 8.3%). Coexistence of extended-spectrum β-lactamase (ESBL) genes and OmpF and/or OmpC loss was found in 27/36 isolates; additionally, increased co-expression of efflux pump genes acrB and oqxA was identified in 25/36 isolates, which may together contribute to co-resistance to carbapenems and tigecycline. Most ST88 strains carried carbapenemases, especially NDM-1. Overexpression of efflux pumps contributed to tigecycline resistance. The presence of carbapenemase and/or ESBL genes and lack of OMPs, but not efflux pump overexpression, may confer carbapenem resistance. Reasonable supervision and management of the epidemic of TCREC will help to stem the transmission of isolates.

Characterization of Carbapenem-Resistant Enterobacteriaceae Cultured From Retail Meat Products, Patients, and Porcine Excrement in China.

The emergence and dissemination of carbapenem-resistant Enterobacteriaceae (CRE) is a growing concern to animal and public health. However, little is known about the spread of CRE in food and livestock and its potential transmission to humans. To identify CRE strains from different origins and sources, 53 isolates were cultured from 760 samples including retail meat products, patients, and porcine excrement. Antimicrobial susceptibility testing was carried out, followed by phylogenetic typing, whole-genome sequencing, broth mating assays, and plasmids analyses. Forty-three Escherichia coli, nine Klebsiella pneumoniae, and one Enterobacter cloacae isolates were identified, each exhibiting multidrug-resistant phenotypes. Genetically, the main sequence types (STs) of E. coli were ST156 (n = 7), ST354 (n = 7), and ST48 (n = 7), and the dominant ST of K. pneumoniae is ST11 (n = 5). blaNDM–5 (n = 40) of E. coli and blaKPC–2 (n = 5) were the key genes that conferred carbapenem resistance phenotypes in these CRE strains. Additionally, the mcr-1 gene was identified in 17 blaNDM-producing isolates. The blaNDM–5 gene from eight strains could be transferred to the recipients via conjugation assays. Two mcr-1 genes in the E. coli isolates could be co-transferred along with the blaNDM–5 genes. IncF and IncX3 plasmids have been found to be predominantly associated with blaNDM gene in these strains. Strains isolated in our study from different sources and regions tend to be concordant and overlap. CRE strains from retail meat products are a reservoir for transition of CRE strains between animals and humans. These data also provide evidence of the dissemination of CRE strains and carbapenem-resistant genes between animal and human sources.

In Vitro Synergism of Azithromycin Combination with Antibiotics against OXA-48-Producing Klebsiella pneumoniae Clinical Isolates.

Carbapenem-resistant Klebsiella pneumoniae has globally emerged as an urgent threat leading to the limitation for treatment. K. pneumoniae carrying blaOXA-48, which plays a broad magnitude of carbapenem susceptibility, is widely concerned. This study aimed to characterize related carbapenem resistance mechanisms and forage for new antibiotic combinations to combat blaOXA-48-carrying K. pneumoniae. Among nine isolates, there were two major clones and a singleton identified by ERIC-PCR. Most isolates were resistant to ertapenem (MIC range: 2–>256 mg/L), but two isolates were susceptible to imipenem and meropenem (MIC range: 0.5–1 mg/L). All blaOXA-48-carrying plasmids conferred carbapenem resistance in Escherichia coli transformants. Two ertapenem-susceptible isolates carried both outer membrane proteins (OMPs), OmpK35 and OmpK36. Lack of at least an OMP was present in imipenem-resistant isolates. We evaluated the in vitro activity of an overlooked antibiotic, azithromycin, in combination with other antibiotics. Remarkably, azithromycin exhibited synergism with colistin and fosfomycin by 88.89% and 77.78%, respectively. Bacterial regrowth occurred after exposure to colistin or azithromycin alone. Interestingly, most isolates were killed, reaching synergism by this combination. In conclusion, the combination of azithromycin and colistin may be an alternative strategy in dealing with blaOXA-48-carrying K. pneumoniae infection during a recent shortage of newly effective antibiotic development.

First Report of bla OXA-677 with Enhanced Meropenem-Hydrolyzing Ability in Pseudomonas aeruginosa in China.

PurposeOXA-10-type class D β-lactamases have shown their evolutionary potential of enhancing carbapenem resistance. This study aimed to elucidate the role of OXA-10 variants in clinical isolated multidrug resistant (MDR) Pseudomonas aeruginosa and characterize the first appearance of OXA-677 in China.MethodsSix blaOXA-10-like-positive strains were screened by PCR from 41 P. aeruginosa strains, which were resistant to both carbapenems and ceftazidime-avibactam, collected across China in 2018. The minimum inhibitory concentrations (MIC) were determined with the broth microdilution method. The resistance-associated genes and genetic environment were investigated by whole-genome sequencing (WGS). The function and mechanism of OXA-677 β-lactamase were identified by molecular cloning and protein structure modeling.ResultsAll the blaOXA-10-like-positive Pseudomonas aeruginosa were MDR strains. They also had outer membrane porin defects and produced β-lactam resistance gene blaPER-1, fluoroquinolone-resistant gene crpP, aminoglycoside-resistance gene aph(3ʹ)-IIb, aph(6)-Id, aacA and aadA, fosfomycin-resistance gene fosA, sulfamethoxazole-resistance gene sul1, and chloramphenicol-resistance gene catB7. All blaOXA-10 variants were located in a Tn1403-related transposon, containing aacA4-12-blaOXA-677-aadA1, aacA4-12-blaOXA-101-aadA5, and blaOXA-246-aacA3-aadA13 gene cassette arrays, respectively. Notably, the blaOXA-677 producer showed a high MIC level of meropenem (MIC>64 mg/L). Compared to blaOXA-10, blaOXA-677 was found a G-to-T transversion at position 350, leading to a phenylalanine-for-valine substitution in position 117, which is closer to leucine155 in the omega loop of the active site. MIC of meropenem for E. coli DH5α with the recombinant plasmid pHSG398 carrying blaOXA-677 was elevated by 8 times.ConclusionWe speculate that the OXA-10-like enzymes and the decrease of membrane permeability confer carbapenem resistance, and the V117 substitution in OXA-677 might lead to a higher resistance level of meropenem.

Subtype Screening of blaIMP Genes Using Bipartite Primers for DNA Sequencing.

Genes conferring carbapenem resistance have spread worldwide among gram-negative bacteria. Subtyping of these genes has epidemiological value due to the global cross-border movement of people. Subtyping of blaIMP genes that frequently detected in Japan appears to be important in public health settings; however, there are few useful tools for this purpose. We developed a subtyping screening tool based on PCR direct sequencing, which targets the internal sequences of almost all blaIMP genes. The tool used bipartite multiplex primers with M13 universal sequences at the 5'-end. According to in silico analysis, among the 78 known IMP-type genes, except for blaIMP-81, 77 detected genes were estimated to be differentiated. In vitro evaluation indicated that sequences of amplicons of IMP-1, IMP-6, IMP-7, and IMP-20 templates were identical to their respective subtypes. Even if the amplicons were small or undetectable through the first PCR, sufficient amplicons for DNA sequencing were obtained through a second PCR using the M13 universal primers. In conclusion, our tool can be possibly used for subtype screening of blaIMP, which is useful for the surveillance of bacteria with blaIMP in clinical and public health settings or environmental fields.

Genomic characterization of triple-carbapenemase-producing Acinetobacter baumannii.

ObjectivesTo characterize Acinetobacter baumannii OCU_Ac16a, a clinical isolate co-harbouring three acquired carbapenemase genes, blaNDM-1, blaTMB-1, and blaOXA-58, and assess the clinical significance of so-called multiple-carbapenemase producers.MethodsOCU_Ac16a and its close relative, OCU_Ac16b, which lacks the blaNDM-1, were isolated from sputum cultures of a patient at Osaka City University Hospital. We subjected these strains to whole-genome analysis, particularly focusing on the genetic context of each carbapenemase gene. The transmissibility and functionality of each carbapenemase gene were analysed by conjugation and transformation experiments and antimicrobial susceptibility tests.Results bla TMB-1 was located in a class 1 integron on the chromosome, whereas blaNDM-1 and blaOXA-58 were found on plasmids named pOCU_Ac16a_2 and pOCU_Ac16a_3, respectively. pOCU_Ac16a_2 (which exhibited highly efficient self-transmissibility) and pOCU_Ac16a_3 (which did not show transmissibility but could be introduced into another A. baumannii strain via electroporation) could both confer carbapenem resistance (MICs ≥512 and ≥32 mg/L, respectively) on the recipient strain. The functionality of blaTMB-1 was evident from the high resistance of OCU_Ac16b to ceftazidime and cefepime (MICs ≥256 and 48 mg/L, respectively), and the high resistance of OCU_Ac16a to cefiderocol (MIC 32 mg/L) could be explained by the additive effect of blaNDM-1 and blaTMB-1.ConclusionsOur data revealed the genomic organization of OCU_Ac16a and demonstrated that all the carbapenemase genes are functional, each contributing to the extremely high broad-spectrum resistance of OCU_Ac16a to β-lactams. As multiple-carbapenemase producers can be serious health threats as drug-resistant pathogens and disseminators of carbapenemase genes, close attention should be paid to their emergence.

Characterization of the novel plasmid-encoded MBL gene blaAFM-1, integrated into a blaIMP-45-bearing transposon Tn6485e in a carbapenem-resistant Pseudomonas aeruginosa clinical isolate.

To characterize the novel subclass B1 MBL AFM-1, encoded by a blaIMP-45-bearing megaplasmid from a carbapenem-resistant Pseudomonas aeruginosa (CRPA) clinical isolate. CRPA HS17-127 and its transconjugant were discovered to carry blaAFM-1 in our previous study. blaAFM-1 and blaNDM-1 were cloned and expressed in Escherichia coli TOP10 and P. aeruginosa PAO1, respectively, to test the resistance phenotype. Kinetic studies were performed to elucidate the biochemical characteristics of the AFM-1 enzyme. Comparative genomic analysis was applied to investigate the genetic context of blaAFM-1. PAO1 transconjugant TcHS17-127 exhibited carbapenem resistance with an imipenem MIC of 64 mg/L. E. coli transformants with cloned blaAFM-1 or blaNDM-1 had increased MICs of all β-lactams tested (except aztreonam) and imipenem MICs of 4-8 mg/L. Kinetic studies showed that AFM-1 had greater catalytic efficiency against cephalosporins than carbapenems. blaAFM-1 was located on a 486 963 bp IncP-2 plasmid, pHS17-127, containing a 57.3 kb MDR Tn1403-derivative transposon, Tn6485e, which is genetically closest to the blaIMP-45-bearing Tn6485 transposon but has acquired an extra ISCR27n3-blaAFM-1 module. Multicentre surveillance of 605 P. aeruginosa clinical isolates identified three blaAFM carriers from different STs. Two of them co-carried blaAFM-1 and blaIMP-45. A BLAST search against the NCBI database showed six blaAFM carriers on various plasmids and the chromosomes of different Gram-negative species. The blaAFM-1 gene confers carbapenem resistance and has been captured in distinct species of non-fermenters. Co-carriage of blaAFM-1 and blaIMP-45 in an MDR transposon on a conjugative plasmid can be expected to promote further dissemination of blaMBLs.

Detection of IncN-pST15 one-health plasmid harbouring blaKPC-2 in a hypermucoviscous Klebsiella pneumoniae CG258 isolated from an infected dog, Brazil.

The emergence and rapid spread of carbapenemase‐producing Enterobacterales represents a serious public health concern. Critically, these global priority bacteria have begun to be reported in companion animals, implying a potential risk of cross‐transmission between humans and pets. Using long‐read (MinION) and short‐read (Illumina) sequencing technologies, we have identified and characterized a hypermucoviscous KPC‐2‐producing Klebsiella pneumoniae strain belonging to the high‐risk international clone ST11/CG258, in a dog with urinary tract infection. Strikingly, the bla KPC‐2 gene was carried by a 54‐kb IncN plasmid assignated to ST15, which shared 99.8 and 96.8% pairwise identity with IncN‐pST15 plasmids from human and environmental K. pneumoniae strains, respectively; all come from an area with high endemicity of KPC‐2. Our findings suggest that IncN‐pST15 plasmids conferring carbapenem resistance can play as important a role as clonal transmission of K. pneumoniae, representing another major challenge for One Health.

Variability in carbapenemase activity of intrinsic OxaAb (OXA-51-like) β-lactamase enzymes in Acinetobacter baumannii.

To measure the variability in carbapenem susceptibility conferred by different OxaAb variants, characterize the molecular evolution of oxaAb and elucidate the contribution of OxaAb and other possible carbapenem resistance factors in the clinical isolates using WGS and LC-MS/MS. Antimicrobial susceptibility tests were performed on 10 clinical Acinetobacter baumannii isolates. Carbapenem MICs were evaluated for all oxaAb variants cloned into A. baumannii CIP70.10 and BM4547, with and without their natural promoters. Molecular evolution analysis of the oxaAb variants was performed using FastTree and SplitsTree4. Resistance determinants were studied in the clinical isolates using WGS and LC-MS/MS. Only the OxaAb variants with I129L and L167V substitutions, OxaAb(82), OxaAb(83), OxaAb(107) and OxaAb(110) increased carbapenem MICs when expressed in susceptible A. baumannii backgrounds without an upstream IS element. Carbapenem resistance was conferred with the addition of their natural upstream ISAba1 promoter. LC-MS/MS analysis on the original clinical isolates confirmed overexpression of the four I129L and L167V variants. No other differences in expression levels of proteins commonly associated with carbapenem resistance were detected. Elevated carbapenem MICs were observed by expression of OxaAb variants carrying clinically prevalent substitutions I129L and L167V. To drive carbapenem resistance, these variants required overexpression by their upstream ISAba1 promoter. This study clearly demonstrates that a combination of IS-driven overexpression of oxaAb and the presence of particular amino acid substitutions in the active site to improve carbapenem capture is key in conferring carbapenem resistance in A. baumannii and other mechanisms are not required.