Conferring Carbapenem Resistance Research Articles

P-1347. Characterizing Atypical Antibiotic Non-Susceptibility Features of Emergent U.S. Carbapenem-resistant Acinetobacter baumannii Lineages

Abstract Background Due to elevated genetic divergence between different Carbapenem-resistant (CR) A. baumannii (Ab) lineages, CRAb isolates belonging to clonal groups (CGs) currently emerging in the U.S. may display traits that differ from those of unrelated international CGs. Variations in CG-associated physiology could especially affect the choice and interpretation of antimicrobial susceptibility tests (AST) for these multidrug resistant lineages. Methods We integrated multimodal AST and comparative genomic analysis of CG108, CG406, and CG499 isolates identified in two U.S. cities since 2017. Lineage-specific resistance features were queried among genomes of isolates from other U.S. locations. We further investigated a unique sulfonamide (SXT) heteroresistance trait via mutational analysis. Results Most antibiotic non-susceptibility was attributable to conventional, lineage-independent resistance determinants on mobile genetic elements. In contrast, a FtsI A515V polymorphism shared among CG108 genomes appears to confer carbapenem resistance independent of carbapenamases, while genomes of various CG406 CRAb isolates lacked any readily identifiable CR determinant. We observed antibiotic-specific heteroresistance against sulbactam (SAM) and imipenem among CG108 isolates and against SXT among CG499 isolates. Integrity of the wzc gene in a CG499 isolate was necessary for high-molecular weight capsule polysaccharide (KPS) production and SXT heteroresistance. Despite lacking any conventional resistance determinants, the wzc-deficient mutant and all other CG499 isolates displayed greater resistance to SXT compared to most susceptible Ab isolates. Conclusion We describe AST features unique to emerging CRAb CGs and independent of conventional extrinsic resistance genes. CG499 SXT heteroresistance appears to rely on the ability to produce high-molecular weight KPS, though another unidentified mechanism also contributes to decreased susceptibility compared to wild-type. We propose ongoing surveillance efforts must recognize lineage-related variations to best tailor new strategies against the evolving nature of CRAb disease. Disclosures Megan Amerson-Brown, PhD, Selux diagnostics: Grant/Research Support

Colistin, doxycycline and Labetalol-meropenem combination are the most active against XDR-Carbapenem-resistant Acinetobacter baumannii: Role of a novel transferrable plasmid conferring carbapenem resistance

This study aimed to evaluate the antimicrobial susceptibility and combination of a beta-blocker, labetalol (LAB) and meropenem (MEM) on Carbapenem-resistant (CR) A. baumannii clinical isolates. A total of 43 CR- A. baumannii were isolated of which 37 (86.6 %) and 28 (65 %) exhibited MDR and XDR phenotypes, respectively. Colistin and doxycycline still retain their activities in 93.1 % and 72.1 % of the isolates, respectively. Combining MEM with LAB at 0.25 mg /mL, decreased MIC values in 91.4 % (32/35) however, at 0.5 mg /mL, it decreased MIC value and restored susceptibility to MEM in 100 % and 91.4 % of the tested isolates, respectively. A novel transferable plasmid pAcbGIM3 harboring aph-3′, blaoxa-58,blaGIM3 and blaCTX-M3 and eight mobile genetic elements were successfully isolated from a pan-drug resistant (PDR) isolate. In conclusion, LAB-MEM is a promising combination and should be clinically examined. This is the first report of a transmissible plasmid harboring blaGIM3 gene in Egypt.

Investigation on the mechanisms of carbapenem resistance among the non-carbapenemase-producing carbapenem-resistant Klebsiella pneumoniae.

In Malaysia, an increase in non-carbapenemase-producing carbapenem-resistant Klebsiella pneumoniae (NC-CRKP) has been observed over the years. Previously, four NC-CRKP with increased susceptibility to ciprofloxacin in the presence of phenylalanine-arginine β-naphthylamide (PAβN) were identified. However, no contribution of the PAβN-inhibited efflux pump to carbapenem resistance was observed. All four NC-CRKP harboured non-carbapenemase β-lactamase, with two also exhibiting porin loss. In this study, we further investigated the genomic features and resistance mechanisms of these four isolates. All four NC-CRKP were subjected to whole-genome sequencing, followed by comparative genomic and phylogenetic analyses. Multi-locus sequence typing (MLST) analysis divided the four NC-CRKP into different sequence types: ST392, ST45, ST14, and ST5947. Neither major nor rare carbapenemase genes were detected. Given the presence of non-carbapenemase β-lactamase in all isolates, we further investigated the potential mechanisms of resistance by identifying related chromosomal mutations. Deletion mutation was detected in the cation efflux system protein CusF. Insertion mutation was identified in the nickel/cobalt efflux protein RcnA. Missense mutation of ompK36 porin was detected in two isolates, while the loss of ompK36 porin was observed in another two isolates. This study revealed that NC-CRKP may confer carbapenem resistance through a combination of non-carbapenemase β-lactamase and potential chromosomal mutations including missense mutation or loss of ompK36 porin and/or a frameshift missense mutation in efflux pump systems, such as cation efflux system protein CusF and nickel/cobalt efflux protein RcnA. Our findings highlighted the significance of implementing whole-genome sequencing into clinical practice to promote the surveillance of carbapenem resistance mechanisms among NC-CRKP.

The presence of genes encoding carbapenem-hydrolyzing oxacillinase and lack of carbapenem resistance in non-baumannii Acinetobacter misidentified as Acinetobacter baumannii causing bloodstream infections in a tertiary hospital over a 3-year period

ObjectiveThis study aims to analyze the genomic and clinical characteristics of Non-baumannii Acinetobacter strains misidentified as A. baumannii, causing bloodstream infections (BSIs) in our hospital. Materials and methodsWhole genome sequencing was performed and average nucleotide identity (ANI) was analyzed. Susceptibility testing was conducted using micro-broth methods. The distribution of antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) was examined using online software tools. The prevalence of virulence factors (VFs) was investigated through nucleotide coding sequence comparisons. Genetic structures of blaOXA genes were analyzed by Gcluster software. Clinical information was collected from electronic medical records for patient characterization. ResultsANI analysis identified five strains as Acinetobacter pittii, with the remaining four identified as A. geminorum, A. nosocomialis, A. soli and A. bereziniae. The GC content of all isolates was less than 38.9 % except for A. soli 16,294. All Non-baumannii Acinetobacter strains were relatively susceptible to antibiotics, except for one A. pittii isolate. Nine blaOXA variants were identified in seven isolates, with two isolates co-carrying 2 different types of blaOXA. Twenty-four insertion sequences (ISs) were identified, with ISAba and IS17 being the primary ISs. Five A. pittii isolates shared the same genetic structures around blaOXA. Genes related to adherence, immune modulation, and nutritional/metabolic factors were the most frequent. Few VFs were detected in A. soli 16,294 and A.bereziniae 14,325. ConclusionsThe presence of carbapenem hydrolyzing oxacillinase encoding genes did not confer carbapenem resistance, possibly due to the lack of ISs in the blaOXA flanking sequences. Different blaOXA variants within distinct strains shared the same genetic structures, suggesting potential for multidrug resistance development, which warrants our attention.

Genomic characterisation of a multidrug-resistant Klebsiella pneumoniae co-harbouring blaNDM-1, blaKPC-2, and tet(A) isolated from the bloodstream infections of patients

Carbapenem-resistant Klebsiella pneumoniae (CRKP), a superbug that can be difficult or impossible to treat, has become a worldwide problem. This study presents the first report of a CRKP strain carrying a plasmid co-harbouring blaNDM-1, blaKPC-2, and tet(A) and the subsequent analysis of its genomic features. Isolation and identification of bacteria, antimicrobial susceptibility test, whole genome sequencing, and conjugation experiments assay were conducted in clinical epidemiological investigations and plasmid genetic characterisation analysis. A total of 116 strains of bacteria were isolated from patients with bloodstream infections (BSI) between 2018 and 2023. A total of 89.66% of the isolates were carbapenem-resistant Enterobacteriaceae (CRE), with the majority (75/116) being CRKP. Among these, a novel plasmid co-harbouring blaNDM-1, blaKPC-2, and tet(A) simultaneously was found in CRKP46, and the three genes mediated conjugation by IS26, ISAba125, and IS26, respectively. This plasmid conferred carbapenem resistance to E. coli J53 after conjugative transfer, which was 2 times greater than that of CRKP46. The present study identified the occurrence of a rare plasmid co-harbouring blaNDM-1, blaKPC-2, and tet(A), and the spread of these genes was mediated by the corresponding mobile elements. The increased carbapenem resistance created by this novel plasmid challenges public health security and poses a potential threat to human health; therefore, it deserves attention.

Genomic analysis of carbapenem- and colistin-resistant Klebsiella pneumoniae complex harbouring mcr-8 and mcr-9 from individuals in Thailand

The surge in mobile colistin-resistant genes (mcr) has become an increasing public health concern, especially in carbapenem-resistant Enterobacterales (CRE). Prospective surveillance was conducted to explore the genomic characteristics of clinical CRE isolates harbouring mcr in 2015–2020. In this study, we aimed to examine the genomic characteristics and phonotypes of mcr-8 and mcr-9 harbouring carbapenem-resistant K. pneumoniae complex (CRKpnC). Polymerase chain reaction test and genome analysis identified CRKpnC strain AMR20201034 as K. pneumoniae (CRKP) ST147 and strain AMR20200784 as K. quasipneumoniae (CRKQ) ST476, harbouring mcr-8 and mcr-9, respectively. CRKQ exhibited substitutions in chromosomal-mediated colistin resistance genes (pmrB, pmrC, ramA, and lpxM), while CRKP showed two substitutions in crrB, pmrB, pmrC, lpxM and lapB. Both species showed resistance to colistin, with minimal inhibitory concentrations of 8 µg/ml for mcr-8-carrying CRKP isolate and 32 µg/ml for mcr-9-carrying CRKQ isolate. In addition, CRKP harbouring mcr-8 carried blaNDM, while CRKQ harbouring mcr-9 carried blaIMP, conferring carbapenem resistance. Analysis of plasmid replicon types carrying mcr-8 and mcr-9 showed FIA-FII (96,575 bp) and FIB-HI1B (287,118 bp), respectively. In contrast with the plasmid carrying the carbapenemase genes, the CRKQ carried blaIMP-14 on an IncC plasmid, while the CRKP harboured blaNDM-1 on an FIB plasmid. This finding provides a comprehensive insight into another mcr-carrying CRE from patients in Thailand. The other antimicrobial-resistant genes in the CRKP were blaCTX-M-15, blaSHV-11, blaOXA-1, aac(6′)-Ib-cr, aph(3′)-VI, ARR-3, qnrS1, oqxA, oqxB, sul1, catB3, fosA, and qacE, while those detected in CRKQ were blaOKP-B-15, qnrA1, oqxA, oqxB, sul1, fosA, and qacE. This observation highlights the importance of strengthening official active surveillance efforts to detect, control, and prevent mcr-harbouring CRE and the need for rational drug use in all sectors.

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.

Emergence of Carbapenem-resistant Clinical Isolates of Providencia Species.

Providencia is a genus of Gram-negative and non-spore forming bacteria belonging to the family Morganellaceae, which causes opportunistic infections in humans. Of the 10 Providencia species identified to date, three, P. alcalifaciens, P. rettgeri and P. stuartii, are clinically important. P. alcalifaciens causes diarrhea, including outbreaks arising from food-borne infections, and P. stuartii and P. rettgeri have been found to cause hospital acquired urinary tract infections. Four isolates of P. rettgeri and one isolate of P. stuartii were obtained from urine samples of five patients in Japan in 2018. All five isolates were highly resistant to carbapenems. Three isolates harbored bla IMP-70, encoding a variant of IMP-1 metallo-β-lactamase, with two amino acid substitutions (Val67Phe and Phe87Val), one isolate harbored two copies of bla IMP-1 and one isolate harbored bla IMP-11. Expression of bla IMP-70 conferred carbapenem resistance in Escherichia coli. Recombinant IMP-10, an IMP-1 variant with Val67Phe but without Phe87Val, had significant higher hydrolytic activities against meropenem than recombinant IMP-1, indicating that the Val67Phe amino acid substitution alters activities against meropenem in IMP-70. These results suggest that Providencia species. become more highly resistant to carbapenems by acquisition of two copies of bla IMP-1 or by mutations in bla IMP that result in amino acid substitutions, such as bla IMP-70.

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.