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153500.9919Complete Genome Sequencing of Acinetobacter baumannii AC1633 and Acinetobacter nosocomialis AC1530 Unveils a Large Multidrug-Resistant Plasmid Encoding the NDM-1 and OXA-58 Carbapenemases. Carbapenem-resistant Acinetobacter spp. are considered priority drug-resistant human-pathogenic bacteria. The genomes of two carbapenem-resistant Acinetobacter spp. clinical isolates obtained from the same tertiary hospital in Terengganu, Malaysia, namely, A. baumannii AC1633 and A. nosocomialis AC1530, were sequenced. Both isolates were found to harbor the carbapenemase genes bla(NDM-1) and bla(OXA-58) in a large (ca. 170 kb) plasmid designated pAC1633-1 and pAC1530, respectively, that also encodes genes that confer resistance to aminoglycosides, sulfonamides, and macrolides. The two plasmids were almost identical except for the insertion of ISAba11 and an IS4 family element in pAC1633-1, and ISAba11 along with relBE toxin-antitoxin genes flanked by inversely orientated pdif (XerC/XerD) recombination sites in pAC1530. The bla(NDM-1) gene was encoded in a Tn125 composite transposon structure flanked by ISAba125, whereas bla(OXA-58) was flanked by ISAba11 and ISAba3 downstream and a partial ISAba3 element upstream within a pdif module. The presence of conjugative genes in plasmids pAC1633-1/pAC1530 and their discovery in two distinct species of Acinetobacter from the same hospital are suggestive of conjugative transfer, but mating experiments failed to demonstrate transmissibility under standard laboratory conditions. Comparative sequence analysis strongly inferred that pAC1633-1/pAC1530 was derived from two separate plasmids in an IS1006-mediated recombination or transposition event. A. baumannii AC1633 also harbored three other plasmids designated pAC1633-2, pAC1633-3, and pAC1633-4. Both pAC1633-3 and pAC1633-4 are cryptic plasmids, whereas pAC1633-2 is a 12,651-bp plasmid of the GR8/GR23 Rep3-superfamily group that encodes the tetA(39) tetracycline resistance determinant in a pdif module.IMPORTANCE Bacteria of the genus Acinetobacter are important hospital-acquired pathogens, with carbapenem-resistant A. baumannii listed by the World Health Organization as the one of the top priority pathogens. Whole-genome sequencing of carbapenem-resistant A. baumannii AC1633 and A. nosocomialis AC1530, which were isolated from the main tertiary hospital in Terengganu, Malaysia, led to the discovery of a large, ca. 170-kb plasmid that harbored genes encoding the New Delhi metallo-β-lactamase-1 (NDM-1) and OXA-58 carbapenemases alongside genes that conferred resistance to aminoglycosides, macrolides, and sulfonamides. The plasmid was a patchwork of multiple mobile genetic elements and comparative sequence analysis indicated that it may have been derived from two separate plasmids through an IS1006-mediated recombination or transposition event. The presence of such a potentially transmissible plasmid encoding resistance to multiple antimicrobials warrants vigilance, as its spread to susceptible strains would lead to increasing incidences of antimicrobial resistance.202133504662
153810.9916KPC-2 allelic variants in Klebsiella pneumoniae isolates resistant to ceftazidime-avibactam from Argentina: bla(KPC-80), bla(KPC-81), bla(KPC-96) and bla(KPC-97). Ceftazidime-avibactam (CZA) therapy has significantly improved survival rates for patients infected by carbapenem-resistant bacteria, including KPC producers. However, resistance to CZA is a growing concern, attributed to multiple mechanisms. In this study, we characterized four clinical CZA-resistant Klebsiella pneumoniae isolates obtained between July 2019 and December 2020. These isolates expressed novel allelic variants of bla(KPC-2) resulting from changes in hotspots of the mature protein, particularly in loops surrounding the active site of KPC. Notably, KPC-80 had an K269_D270insPNK mutation near the Lys270-loop, KPC-81 had a del_I173 mutation within the Ω-loop, KPC-96 showed a Y241N substitution within the Val240-loop and KPC-97 had an V277_I278insNSEAV mutation within the Lys270-loop. Three of the four isolates exhibited low-level resistance to imipenem (4 µg/mL), while all remained susceptible to meropenem. Avibactam and relebactam effectively restored carbapenem susceptibility in resistant isolates. Cloning mutant bla(KPC) genes into pMBLe increased imipenem MICs in recipient Escherichia coli TOP10 for bla(KPC-80), bla(KPC-96), and bla(KPC-97) by two dilutions; again, these MICs were restored by avibactam and relebactam. Frameshift mutations disrupted ompK35 in three isolates. Additional resistance genes, including bla(TEM-1), bla(OXA-18) and bla(OXA-1), were also identified. Interestingly, three isolates belonged to clonal complex 11 (ST258 and ST11) and one to ST629. This study highlights the emergence of CZA resistance including unique allelic variants of bla(KPC-2) and impermeability. Comprehensive epidemiological surveillance and in-depth molecular studies are imperative for understanding and monitoring these complex resistance mechanisms, crucial for effective antimicrobial treatment strategies. IMPORTANCE: The emergence of ceftazidime-avibactam (CZA) resistance poses a significant threat to the efficacy of this life-saving therapy against carbapenem-resistant bacteria, particularly Klebsiella pneumoniae-producing KPC enzymes. This study investigates four clinical isolates exhibiting resistance to CZA, revealing novel allelic variants of the key resistance gene, bla(KPC-2). The mutations identified in hotspots surrounding the active site of KPC, such as K269_D270insPNK, del_I173, Y241N and V277_I278insNSEAV, prove the adaptability of these pathogens. Intriguingly, low-level resistance to imipenem and disruptions in porin genes were observed, emphasizing the complexity of the resistance mechanisms. Interestingly, three of four isolates belonged to clonal complex 11. This research not only sheds light on the clinical significance of CZA resistance but also shows the urgency for comprehensive surveillance and molecular studies to inform effective antimicrobial treatment strategies in the face of evolving bacterial resistance.202438319084
121120.9914Molecular characterization of multidrug-resistant Escherichia coli of the phylogroups A and C in dairy calves with meningitis and septicemia. Escherichia coli is an important cause of septicemia (SEPEC) and neonatal meningitis (NMEC) in dairy calves. However, the diversity of virulence profiles, phylogroups, antimicrobial resistance patterns, carriage of integron structures, and fluoroquinolone (FQ) resistance mechanisms have not been fully investigated. Also, there is a paucity of knowledge about the virulence profiles and frequency of potential SEPEC in feces from calves with or without diarrhea. This study aimed to characterize the virulence potential, phylogroups, antimicrobial susceptibility, integron content, and FQ-resistance mechanisms in Escherichia coli isolated from calves with meningitis and septicemia. Additionally, the virulence genes (VGs) and profiles of E. coli isolated from diarrheic and non-diarrheic calves were compared between them and together with NMEC and SEPEC in order to identify shared profiles. Tissue and fluid samples from eight dairy calves with septicemia, four of which had concurrent meningitis, were processed for bacteriology and histopathology. Typing of VGs was assessed in 166 isolates from diverse samples of each calf. Selected isolates were evaluated for antimicrobial susceptibility by the disk diffusion test. Phylogroups, integron gene cassettes cartography, and FQ-resistance determinants were analyzed by PCR, sequencing, and bioinformatic tools. Furthermore, 109 fecal samples and 700 fecal isolates from dairy calves with or without diarrhea were evaluated to detect 19 VGs by uniplex PCR. Highly diverse VG profiles were characterized among NMEC and SEPEC isolates, but iucD was the predominant virulence marker. Histologic lesions in all calves supported their pathogenicity. Selected isolates mainly belonged to phylogroups A and C and showed multidrug resistance. Classic (dfrA17 and arr3-dfrA27) and complex (dfrA17-aadA5::ISCR1::bla(CTX-M-2)) class 1 integrons were identified. Target-site mutations in GyrA (S83L and D87N) and ParC (S80I) encoding genes were associated with FQ resistance. The VGs detected more frequently in fecal samples included f17G (50%), papC (30%), iucD (20%), clpG (19%), eae (16%), and afaE-8 (13%). Fecal isolates displaying the profiles of f17 or potential SEPEC were found in 25% of calves with and without diarrhea. The frequency of E. coli VGs and profiles did not differ between both groups (p > 0.05) and were identical or similar to those found in NMEC and SEPEC. Overall, multidrug-resistant E. coli isolates with diverse VG profiles and belonging to phylogroups A and C can be implicated in natural cases of meningitis and septicemia. Their resistance phenotypes can be partially explained by class 1 integron gene cassettes and target-site mutations in gyrA and parC. These results highlight the value of antimicrobial resistance surveillance in pathogenic bacteria isolated from food-producing animals. Besides, calves frequently shed potential SEPEC in their feces as commensals ("Trojan horse"). Thus, these bacteria may be disseminated in the farm environment, causing septicemia and meningitis under predisposing factors.202234982979
520030.9914Whole genome sequencing of the multidrug-resistant Chryseobacterium indologenes isolated from a patient in Brazil. Chryseobacterium indologenes is a non-glucose-fermenting Gram-negative bacillus. This emerging multidrug resistant opportunistic nosocomial pathogen can cause severe infections in neonates and immunocompromised patients. This study aimed to present the first detailed draft genome sequence of a multidrug-resistant C. indologenes strain isolated from the cerebrospinal fluid of an infant hospitalized at the Neonatal Intensive Care Unit of Brazilian Tertiary Hospital. We first analyzed the susceptibility of C. indologenes strain to different antibiotics using the VITEK 2 system. The strain demonstrated an outstanding resistance to all the antibiotic classes tested, including β-lactams, aminoglycosides, glycylcycline, and polymyxin. Next, C. indologenes was whole-genome-sequenced, annotated using Prokka and Rapid Annotation using Subsystems Technology (RAST), and screened for orthologous groups (EggNOG), gene ontology (GO), resistance genes, virulence genes, and mobile genetic elements using different software tools. The draft genome contained one circular chromosome of 4,836,765 bp with 37.32% GC content. The genomic features of the chromosome present numerous genes related to cellular processes that are essential to bacteria. The MDR C. indologenes revealed the presence of genes that corresponded to the resistance phenotypes, including genes to β-lactamases (bla (IND-13), bla (CIA-3), bla (TEM-116), bla (OXA-209), bla (VEB-15)), quinolone (mcbG), tigecycline (tet(X6)), and genes encoding efflux pumps which confer resistance to aminoglycosides (RanA/RanB), and colistin (HlyD/TolC). Amino acid substitutions related to quinolone resistance were observed in GyrA (S83Y) and GyrB (L425I and K473R). A mutation that may play a role in the development of colistin resistance was detected in lpxA (G68D). Chryseobacterium indologenes isolate harbored 19 virulence factors, most of which were involved in infection pathways. We identified 13 Genomic Islands (GIs) and some elements associated with one integrative and conjugative element (ICEs). Other elements linked to mobile genetic elements (MGEs), such as insertion sequence (ISEIsp1), transposon (Tn5393), and integron (In31), were also present in the C. indologenes genome. Although plasmids were not detected, a ColRNAI replicon type and the most resistance genes detected in singletons were identified in unaligned scaffolds. We provided a wide range of information toward the understanding of the genomic diversity of C. indologenes, which can contribute to controlling the evolution and dissemination of this pathogen in healthcare settings.202235966843
142540.9914Distribution and Antimicrobial Resistance of Complicated Intraabdominal Infection Pathogens in Two Tertiary Hospitals in Egypt. Background: Management of complicated intraabdominal infections (cIAIs) requires containment of the source and appropriate initial antimicrobial therapy. Identifying the local data is important to guide the empirical selection of antimicrobial therapy. In this study, we aimed to describe the pathogen distribution and antimicrobial resistance of cIAI. Methods: In two major tertiary care hospitals in Egypt, we enrolled patients who met the case definition of cIAI from October 2022 to September 2023. Blood cultures were performed using the BACTAlert system (BioMerieux, Marcy l'Etoile, France). A culture of aspirated fluid, resected material, or debridement of the infection site was performed. Identification of pathogens and antimicrobial susceptibility testing were conducted by the VITEK-2 system (BioMerieux, Marcy l'Etoile, France). Gram-negative resistance genes were identified by PCR and confirmed by whole bacterial genome sequencing using the Nextera XT DNA Library Preparation Kit and sequencing with the MiSeq Reagent Kit 600 v3 (Illumina, USA) on the Illumina MiSeq. Results: We enrolled 423 patients, 275 (65.01%) males. The median age was 61.35 (range 25-72 years). We studied 452 recovered bacterial isolates. Gram-negative bacteria were the vast majority, dominated by E. coli, followed by Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Proteus mirabilis (33.6%, 30.5%, 13.7%, 13%, and 5.4%, respectively). High rates of resistance were detected to third- and fourth-generation cephalosporins and fluoroquinolones. No resistance was detected to colistin. Resistance to amikacin and tigecycline was low among all isolates. Resistance to meropenem and ceftazidime/avibactam was moderate. ESBL genes were common in E. coli and K. pneumoniae. CTX-M15 gene was the most frequent. Among Enterobacterales, bla(OXA-48) and bla(NDM) were the most prevalent carbapenemase genes. Pseudomonas aeruginosa isolates harbored a wide variety of carbapenemase genes (OXA, NDM, VIM, SIM, GIM, SPM, IMP, AIM), dominated by metallo-beta-lactamases. In 20.6% of isolates, we identified two or more resistance genes. Conclusion: High resistance rates were detected to third- and fourth-generation cephalosporins and fluoroquinolones. Amikacin and tigecyclines were the most active antimicrobials. Our data call for urgent implementation of antimicrobial stewardship programs and reinforcement of infection control.202439172656
144250.9914Superbugs in the supermarket? Assessing the rate of contamination with third-generation cephalosporin-resistant gram-negative bacteria in fresh Australian pork and chicken. BACKGROUND: Antibiotic misuse in food-producing animals is potentially associated with human acquisition of multidrug-resistant (MDR; resistance to ≥ 3 drug classes) bacteria via the food chain. We aimed to determine if MDR Gram-negative (GNB) organisms are present in fresh Australian chicken and pork products. METHODS: We sampled raw, chicken drumsticks (CD) and pork ribs (PR) from 30 local supermarkets/butchers across Melbourne on two occasions. Specimens were sub-cultured onto selective media for third-generation cephalosporin-resistant (3GCR) GNBs, with species identification and antibiotic susceptibility determined for all unique colonies. Isolates were assessed by PCR for SHV, TEM, CTX-M, AmpC and carbapenemase genes (encoding IMP, VIM, KPC, OXA-48, NDM). RESULTS: From 120 specimens (60 CD, 60 PR), 112 (93%) grew a 3GCR-GNB (n = 164 isolates; 86 CD, 78 PR); common species were Acinetobacter baumannii (37%), Pseudomonas aeruginosa (13%) and Serratia fonticola (12%), but only one E. coli isolate. Fifty-nine (36%) had evidence of 3GCR alone, 93/163 (57%) displayed 3GCR plus resistance to one additional antibiotic class, and 9/163 (6%) were 3GCR plus resistance to two additional classes. Of 158 DNA specimens, all were negative for ESBL/carbapenemase genes, except 23 (15%) which were positive for AmpC, with 22/23 considered to be inherently chromosomal, but the sole E. coli isolate contained a plasmid-mediated CMY-2 AmpC. CONCLUSIONS: We found low rates of MDR-GNBs in Australian chicken and pork meat, but potential 3GCR-GNBs are common (93% specimens). Testing programs that only assess for E. coli are likely to severely underestimate the diversity of 3GCR organisms in fresh meat.201829484175
144860.9913Molecular characteristics of carbapenem-resistant Acinetobacter spp. from clinical infection samples and fecal survey samples in Southern China. BACKGROUND: Carbapenem resistance among Acinetobacter species has become a life-threatening problem. As a last resort in the treatment of gram-negative bacteria infection, resistance to colistin is also a serious problem. The aim of study was to analyze the mechanism of resistance and perform genotyping of carbapenem-resistant Acinetobacter from clinical infection and fecal survey samples in Southern China. METHODS: One hundred seventy and 74 carbapenem-resistant Acinetobacter were isolated from clinical infection samples and fecal survey samples, respectively. We detected the related genes, including carbapenemase genes (bla(KPC), bla(IMP), bla(SPM), bla(VIM), bla(NDM), bla(OXA-23-like), bla(OXA-24/40-like), bla(OXA-51-like), and bla(OXA-58-like)), colistin resistance-related genes (mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5), a porin gene (carO), efflux pump genes (adeA, adeB, adeC, adeI, adeJ, and adeK), mobile genetic element genes (intI1, intI2, intI3, tnpU, tnp513, IS26, ISAba1, and ISAba125), and the integron variable region. Genotyping was analyzed by enterobacterial repetitive intergenic consensus (ERIC)-PCR and dendrogram cluster analysis. RESULTS: Among the 244 carbapenem-resistant Acinetobacter, the common carbapenemase-positive genes included the following: bla(OXA-51-like), 183 (75.00%); bla(OXA-23-like), 174 (71.30%); bla(NDM-1), 57 (23.40%); and bla(OXA-58-like), 30 (12.30%). The coexistence of mcr-1 and bla(NDM-1) in five strains of A. junii was found for the first time. Eleven distinct carO gene variants were detected in 164 (67.20%) strains, and ten novel variants, which shared 92-99% identity with sequences in the Genbank database, were first reported. Efflux system genes were present in approximately 70% of the isolates; adeABC and adeIJK were observed in 76.23 and 72.13%, respectively. Class 1 integrons were detected in 180 (73.80%) strains and revealed that four gene cassette arrays contained 11 distinct genes. The genotyping by ERIC-PCR demonstrated a high genetic diversity of non-baumannii Acinetobacter, and greater than 90% similarity to A. baumannii. CONCLUSIONS: The bla(NDM-1) gene was identified in up to 77% of the carbapenem-resistant Acinetobacter isolated from fecal survey samples, indicating that the gut might be a reservoir of resistant opportunistic bacteria. Intestinal bacteria can be transmitted through the fecal-hand, which is a clinical threat, thus, the monitoring of carbapenem-resistant bacteria from inpatients' feces should be improved, especially for patients who have been using antibiotics for a long time.201931660862
210070.9913Prevalence of Bacteria and Antimicrobial Resistance Genes in Hospital Water and Surfaces. Purpose Antimicrobial resistance (AMR) has become a worldwide environmental and public health problem, causing more than 250,000 deaths per year. Unregulated usage, unsafe hospital practices, and misuse in veterinary contribute to the development of multidrug resistance in various bacteria. Hospital water was hypothesized to be a hotspot for AMR transmission because of (1) increased exposure to antibiotic load, (2) poor drainage and sanitation system, (3) interaction between environmental and clinical microbes. The purpose of the research was to assess the biodiversity and AMR in hospital tap waters. Methodology In this study, the microflora of the hospital tap water and hospital surfaces was observed by obtaining water samples from the intensive care unit (ICU), surgical wards, and washrooms. These were processed through membrane filtration and spread on seven different media (Aeromonas Medium, Azide Dextrose Agar, MacConkey Agar, Mannitol Salt Agar, Pseudomonas Cetrimide Agar, Salmonella Shigella Agar, and Thiosulfate Citrate Bile Salts Sucrose Agar). Surface samples were collected from the faucet, basin, and drain and directly spread on the media plates. Isolates were identified using standard bacteriological and biochemical tests. Kirby-Bauer disk diffusion method was performed using 21 antibiotic disks from 10 different antibiotic classes. They included ampicillin (AMP), amoxicillin (AML), piperacillin-tazobactam (TZP), cefipime (FEP), cefoxitin (FOX), ceftazidime (CAZ), ceftriaxone (CRO), imipenem (IMP), meropenem (MEM), ciprofloxacin (CIP), moxifloxacin (MXF), levofloxacin (LEV), amikacin (AK), gentamicin (CN), tigecycline (TGC), aztreonam (ATM), erythromycin (E), clindamycin (DA), rifampicin (RD), colistin (CT), and chloramphenicol (C). The results were interpreted according to EUCAST guidelines for the antibiogram of the isolates; 38 isolates were selected out of 162 based on different parameters for genotyping and detection of six beta-lactamase genes (blaSHV, blaTEM, blaCTX-M, blaOXA, blaKPC, blaNDM). Results Among these 162 isolates, 82 were obtained from water sources and 80 were collected from surfaces (faucet, basin, drain). The isolates included a variety of bacteria including Aeromonas spp. (20%), Klebsiella spp. (13%), Staphylococcus aureus (13%), Pseudomonas spp.(10%), Escherichia coli (9%), Vibrio spp. (8%), Enterococcus spp. (6%), Shigella spp. (6%), Salmonella spp. (4%), Acinetobacter spp. (3%), Staphylococcus epidermitis (3%), Streptococci spp. (2%), Proteus spp. (1%), Citrobacter spp. (1%), and Serratia spp. (1%). A diverse range of microbes were identified including clinically relevant bacteria, which shows that the urban water cycle is already contaminated with multidrug-resistant microflora of the hospital settings. Macrolide and lincosamide showed the highest resistance followed by penicillin, monobactam, and cephalosporins. blaSHV and blaTEM were prevalent in samples. blaNDM was also found which manifests as a real threat since it causes resistance against carbapenems and colistin, antibiotics reserved as a last resort against infections. Conclusions This study presented the ground reality of antibiotic resistance in Pakistan and how its subsequent spread poses a great threat to the strides made in the field of medicine and public health. Strict regulations regarding antibiotic usage, hospital effluent, and urban water sanitation must be imposed to curb the devastating effects of this increasing phenomenon.202134790487
118980.9913Detection of the carbapenemase gene bla(VIM-5) in members of the Pseudomonas putida group isolated from polluted Nigerian wetlands. There are increasing concerns about possible dissemination of clinically relevant antibiotic resistance genes, including genes encoding for carbapenemases in the environment. However, little is known about environmental distribution of antibiotic resistance in Africa. In this study, four polluted urban wetlands in Nigeria were investigated as potential reservoirs of carbapenem-resistant bacteria (CRB). CRB were isolated from the wetlands, characterized by Blue-Carba test, MIC determinations and whole genome sequencing (WGS). Nine of 65 bacterial isolates identified as members of the Pseudomonas putida group (P. plecoglossicida and P. guariconensis, respectively) harboured the metallo-beta-lactamase gene bla(VIM-5). WGS revealed the bla(VIM-5) in three novel Tn402-like class 1 integron structures containing the cassette arrays aadB|bla(VIM-5)|bla(PSE-1), aadB|bla(VIM-5)|aadB|bla(PSE-1), and bla(VIM-5)|aadB|tnpA|bla(PSE-1)|smr2|tnpA, respectively. Strains carrying the aadB|bla(VIM-5)|bla(PSE-1) cassette also carried an identical integron without bla(VIM-5). In addition(,) the strains harboured another Tn402-like class 1 integron carrying bcr2, several multidrug resistance efflux pumps, and at least one of ampC, aph(3")-lb, aph(6)-ld, tetB, tetC, tetG, floR, and macAB. This is the first report of a carbapenemase gene in bacteria from environmental sources in Nigeria and the first report of bla(VIM-5) in environmental bacteria isolates. This result underscores the role of the Nigerian environment as reservoir of bacteria carrying clinically relevant antibiotic resistance genes.201830310126
144390.9913Wastewater Surveillance Detected Carbapenemase Enzymes in Clinically Relevant Gram-Negative Bacteria in Helsinki, Finland; 2011-2012. Antimicrobial resistance profiling of pathogens helps to identify the emergence of rare or new resistance threats and prioritize possible actions to be taken against them. The analysis of wastewater (WW) can reveal the circulation of antimicrobial-resistant bacteria (ARB) and antimicrobial resistance genes (ARG) among the catchment communities. Here, we analyzed WW influent samples to determine the prevalence of carbapenemase genes-carrying Gram-negative bacteria (Carba-GNB) in Helsinki, Finland. This study set important historical reference points from the very early stage of the carbapenemase era, during the period 2011-2012. A total of 405 bacterial isolates grown on CHROMagarKPC (n = 195) and CHROMagarESBL (n = 210) from WW influent samples were collected between October 2011 and August 2012 and were analyzed. The bacterial DNA from the isolates was extracted, and the prevalence of carbapenemases genes bla (KPC), bla (NDM), bla (GES), bla (OXA-48), bla (IMP), bla (IMI), and bla (VIM) were screened with multiplexed PCR. All carbapenemase-positive isolates were identified taxonomically to species or genus level with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The nucleic acid extraction was successful for 399 isolates, of which 59 (14.8%) were found to carry carbapenemase genes. A total of 89.8% of the carbapenemase positive isolates (53 out of 59) were obtained from CHROMagarKPC plates and only 10.2% (six out of 59) were obtained from CHROMagar ESBL plates. Among the Carba-GNB isolates, 86.4% were bla (GES) (51 out of 59), 10.2% were bla (KPC) (six out of 59), and 3.4% were bla (VIM) (two out of 59). The most common carba-gene, bla (GES), was carried by 10 different bacterial species, including Aeromonas spp., Enterobacter spp., and Kluyvera spp.; the bla (KPC) gene was carried by Escherichia coli, Klebsiella pneumoniae, and Kluyvera cryocescens; and the bla (VIM) gene was carried by Aeromonas hydrophila/caviae and Citrobacter amalonaticus. This study emphasizes that wastewater surveillance (WWS) can be an additional tool for monitoring antimicrobial resistance (AMR) at the population level.202235722284
1991100.9912A strain defined as a novel species in the Acinetobacter genus co-harboring chromosomal associated tet(X3) and plasmid associated bla (NDM-1) from a beef cattle farm in Hebei, China. INTRODUCTION: The co-existence phenomenon of antibiotic resistance genes (ARGs), particularly of last-resort antibiotics in multi-drug resistant (MDR) bacteria, is of particular concern in the least studied bacterial species. METHODS: In 2023, strain M2 was isolated from the sludge sample at a commercial bovine farm in Hebei province, China, using a MacConkey plate containing meropenem. PCR amplification and Sanger sequencing verified it co-carrying bla (NDM) and tet(X) genes. It was classified within the Acinetobacter genus by MALDI-TOF-MS and 16S rDNA analyses. Whole-genome sequencing (WGS) was performed on the Oxford Nanopore platform, with species-level identification via ANI and dDDH. Antimicrobial susceptibility testing was performed against 20 antibiotics. Conjugation assays employed the filter-mating method using E. coli J53 and Salmonella LGJ2 as recipients. RESULTS: This strain was confirmed as a novel species of Acinetobacter genus, showing resistance to meropenem, ampicillin, ceftazidime, cefepime, gentamicin, kanamycin, fosfomycin, imipenem, ertapenem, and tetracycline. Despite carrying tet(X3), it remained susceptible to tigecycline, omadacycline, and doxycycline. The genome carried 11 ARG types, multiple metal resistance genes (MRGs), and virulence factor (VF) genes. The bla (NDM-1) was located in a skeleton, ISAba125-bla (NDM-1)-ble (MBL)-trpF, which was carried by an ISAba14-mediated rolling-circle-like structure in pM2-2-NDM-1 (rep_cluster_481). Integrative and conjugative element (ICE) and multiple pdif modules (driven by the XerCD site-specific recombination (XerCD SSR) system), which were associated with the mobilization of resistance determinants, were identified in this plasmid. Chromosomal tet(X3) was mediated by ISVsa3, forming a skeleton, ISVsa3-XerD-tet (X3)-res-ISVsa3. DISCUSSION: The co-occurrence of bla (NDM) and tet(X) in a novel species of the Acinetobacter genus hints that substantial undiscovered bacteria co-carrying high-risk ARGs are concealing in the agroecological system, which should cause particular concern.202540673007
2459110.9912In vitro antimicrobial activity and resistance mechanisms of cefiderocol against clinical carbapenem-resistant gram-negative bacteria. BACKGROUND: The rise of carbapenem-resistant gram-negative bacteria (CRGNB) necessitates new therapeutic options such as cefiderocol. OBJECTIVE: To evaluate the in vitro efficacy of cefiderocol against clinical CRGNB and investigate associated resistance mechanisms. METHODS: A total of 370 CRGNB isolates were analyzed. Minimum inhibitory concentration (MIC) values were determined, and whole genome sequencing, efflux pump inhibition assays, and RT-qPCR were conducted to assess resistance-related mutations, gene loss, and expression changes. RESULTS: Cefiderocol demonstrated potent in vitro activity, with high susceptibility rates in C. freundii (100%), K. pneumoniae (93.3%), and E. hormaechei (92.2%), and notable activity against P. aeruginosa (80.0%) and Escherichia coli (76.8%). Efflux pump inhibition by Carbonyl Cyanide m-Chlorophenyl Hydrazone (CCCP) significantly reduced MICs in resistant strains. Key resistance mechanisms included β-lactamase gene variants (bla (OXA-66), bla (OXA-23), bla (SHV-12)), mutations in envZ, cirA, nuoC, ampC, and loss or altered expression of iron transporter genes (piuA, pirA, fepA). CONCLUSION: Cefiderocol is highly effective against CRGNB; however, resistance may arise through diverse mechanisms, including efflux pump activity. Continued surveillance of emerging resistance is essential to guide its optimal clinical use.202541113641
1852120.9912Genomic and Resistance Epidemiology of Gram-Negative Bacteria in Africa: a Systematic Review and Phylogenomic Analyses from a One Health Perspective. Antibiotic resistance (AR) remains a major threat to public and animal health globally. However, AR ramifications in developing countries are worsened by limited molecular diagnostics, expensive therapeutics, inadequate numbers of skilled clinicians and scientists, and unsanitary environments. The epidemiology of Gram-negative bacteria, their AR genes, and geographical distribution in Africa are described here. Data were extracted and analyzed from English-language articles published between 2015 and December 2019. The genomes and AR genes of the various species, obtained from the Pathosystems Resource Integration Center (PATRIC) and NCBI were analyzed phylogenetically using Randomized Axelerated Maximum Likelihood (RAxML) and annotated with Figtree. The geographic location of resistant clones/clades was mapped manually. Thirty species from 31 countries and 24 genera from 41 countries were analyzed from 146 articles and 3,028 genomes, respectively. Genes mediating resistance to β-lactams (including bla (TEM-1), bla (CTX-M), bla (NDM), bla (IMP), bla (VIM), and bla (OXA-48/181)), fluoroquinolones (oqxAB, qnrA/B/D/S, gyrA/B, and parCE mutations, etc.), aminoglycosides (including armA and rmtC/F), sulfonamides (sul1/2/3), trimethoprim (dfrA), tetracycline [tet(A/B/C/D/G/O/M/39)], colistin (mcr-1), phenicols (catA/B, cmlA), and fosfomycin (fosA) were mostly found in Enterobacter spp. and Klebsiella pneumoniae, and also in Serratia marcescens, Escherichia coli, Salmonella enterica, Pseudomonas, Acinetobacter baumannii, etc., on mostly IncF-type, IncX(3/4), ColRNAI, and IncR plasmids, within IntI1 gene cassettes, insertion sequences, and transposons. Clonal and multiclonal outbreaks and dissemination of resistance genes across species and countries and between humans, animals, plants, and the environment were observed; Escherichia coli ST103, K. pneumoniae ST101, S. enterica ST1/2, and Vibrio cholerae ST69/515 were common strains. Most pathogens were of human origin, and zoonotic transmissions were relatively limited.IMPORTANCE Antibiotic resistance (AR) is one of the major public health threats and challenges to effective containment and treatment of infectious bacterial diseases worldwide. Here, we used different methods to map out the geographical hot spots, sources, and evolutionary epidemiology of AR. Escherichia coli, Klebsiella pneumoniae, Salmonella enterica, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., Neisseria meningitis/gonorrhoeae, Vibrio cholerae, Campylobacter jejuni, etc., were common pathogens shuttling AR genes in Africa. Transmission of the same clones/strains across countries and between animals, humans, plants, and the environment was observed. We recommend Enterobacter spp. or K. pneumoniae as better sentinel species for AR surveillance.202033234606
1120130.9912Occurrence of NDM-1 and VIM-2 Co-Producing Escherichia coli and OprD Alteration in Pseudomonas aeruginosa Isolated from Hospital Environment Samples in Northwestern Tunisia. Hospital environments constitute the main reservoir of multidrug-resistant bacteria. In this study we aimed to investigate the presence of Gram-negative bacteria in one Northwestern Tunisian hospital environment, and characterize the genes involved in bacterial resistance. A total of 152 environmental isolates were collected from various surfaces and isolated using MacConkey medium supplemented with cefotaxime or imipenem, with 81 fermenter bacteria (27 Escherichia coli, and 54 Enterobacter spp., including 46 Enterobacter cloacae), and 71 non-fermenting bacteria (69 Pseudomonas spp., including 54 Pseudomonas aeruginosa, and 2 Stenotrophomonas maltophilia) being identified by the MALDI-TOF-MS method. Antibiotic susceptibility testing was performed by disk diffusion method and E-Test was used to determine MICs for imipenem. Several genes implicated in beta-lactams resistance were characterized by PCR and sequencing. Carbapenem resistance was detected among 12 isolates; nine E. coli (bla(NDM-1) (n = 8); bla(NDM-1) + bla(VIM-2) (n = 1)) and three P. aeruginosa were carbapenem-resistant by loss of OprD porin. The whole-genome sequencing of P. aeruginosa 97H was determined using Illumina MiSeq sequencer, typed ST285, and harbored bla(OXA-494). Other genes were also detected, notably bla(TEM) (n = 23), bla(CTX-M-1) (n = 10) and bla(CTX-M-9) (n = 6). These new epidemiological data imposed new surveillance strategies and strict hygiene rules to decrease the spread of multidrug-resistant bacteria in this area.202134573959
1423140.9911Distribution and molecular characterization of carbapenemase-producing gram-negative bacteria in Henan, China. This study aimed to investigate the epidemiological characteristics and trends over time of carbapenemase-producing (e.g., KPC, NDM, VIM, IMP, and OXA-48) Gram-negative bacteria (CPGNB). Non-duplicated multi-drug resistant Gram-negative bacteria (MDRGNB) were collected from the First Affiliated Hospital of Zhengzhou University from April 2019 to February 2023. Species identification of each isolate was performed using the Vitek2 system and confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry according to the manufacturer's instructions. PCR detected carbapenem resistance genes in the strains, strains carrying carbapenem resistance genes were categorized as CPGNB strains after validation by carbapenem inactivation assay. A total of 5705 non-repetitive MDRGNB isolates belonging to 78 different species were collected during the study period, of which 1918 CPGNB were validated, with the respiratory tract being the primary source of specimens. Epidemiologic statistics showed a significant predominance of ICU-sourced strains compared to other departments. Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa were the significant CPGNB in Henan, and KPC and NDM were the predominant carbapenemases. Carbapenem-resistant infections in Henan Province showed an overall increasing trend, and the carriage of carbapenemase genes by CPGNB has become increasingly prevalent and complicated. The growing prevalence of CPGNB in the post-pandemic era poses a significant challenge to public safety.202438909136
2778150.9911The investigation of antibiotic residues, antibiotic resistance genes and antibiotic-resistant organisms in a drinking water reservoir system in Germany. Between August 2018 and June 2019, a river system in Germany that supplies a drinking water reservoir and is subject to the discharge from two sewage treatment plants was monitored for antibiotic residues via liquid chromatography-tandem mass spectrometry, antibiotic resistance genes (including bla(NDM), bla(VIM), bla(OXA-48), bla(KPC), bla(GIM), bla(SME), bla(IMI), bla(IMP), bla(SPM), bla(SIM), bla(OXA-23), bla(OXA-24), bla(OXA-51), bla(OXA-58), mcr) via qualitative real-time PCR and antibiotic-resistant bacteria [belonging to the ESKAPE-group (Enterococcus faecium, Staphyhlococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter ssp.; with resistance against Carbapenemases, Cephalosporines and Colistin) and Escherichia coli] based on cultivation methods followed by a characterization via MALDI-TOF MS and susceptibility testing applying microdilution. Residues of macrolide antibiotics such as clarithromycin (up to 0.60 μg/L) and residues of sulfamethoxazole (up to 0.40 μg/L) and trimethoprim (up to 0.39 μg/L) were detected downstream of the sewage treatment plants. In addition, no antibiotic residues were detected upstream the respective sewage treatment plants, except for anhydroerythromycin (n = 1, 202031978723
2768160.9911Prevalence and abundance of antibiotic-resistant genes in culturable bacteria inhabiting a non-polar passu glacier, karakorum mountains range, Pakistan. Natural pristine environments including cold habitats are thought to be the potent reservoirs of antibiotic-resistant genes and have been recurrently reported in polar glaciers' native bacteria, nevertheless, their abundance among the non-polar glaciers' inhabitant bacteria is mostly uncharted. Herein we evaluated antibiotic resistance profile, abundance of antibiotic-resistant genes plus class 1, 2, and 3 integron integrases in 65 culturable bacterial isolates retrieved from a non-polar glacier. The 16S rRNA gene sequencing analysis identified predominantly Gram-negative 43 (66.15%) and Gram-positive 22 (33.84%) isolates. Among the Gram-negative bacteria, Gammaproteobacteria were dominant (62.79%), followed by Betaproteobacteria (18.60%) and Alphaproteobacteria (9.30%), whereas Phyla Actinobacteria (50%) and Firmicutes (40.90%) were predominant among Gram-positive. The Kirby Bauer disc diffusion method evaluated significant antibiotic resistance among the isolates. PCR amplification revealed phylum Proteobacteria predominantly carrying 21 disparate antibiotic-resistant genes like; (bla)AmpC 6 (100%), (bla)VIM-1, (bla)SHV and (bla)DHA 5 (100%) each, (bla)OXA-1 1 (100%), (bla)CMY-4 4 (100%), followed by Actinobacteria 14, Firmicutes 13 and Bacteroidetes 11. Tested isolates were negative for (bla)KPC, qnrA, vanA, ermA, ermB, intl2, and intl3. Predominant Gram-negative isolates had higher MAR index values, compared to Gram-positive. Alignment of protein homology sequences of antibiotic-resistant genes with references revealed amino acid variations in (bla)NDM-1, (bla)OXA-1, (bla)SHV, mecA, aac(6)-Ib3, tetA, tetB, sul2, qnrB, gyrA, and intI1. Promising antibiotic-resistant bacteria, harbored with numerous antibiotic-resistant genes and class 1 integron integrase with some amino acid variations detected, accentuating the mandatory focus to evaluate the intricate transcriptome analysis of glaciated bacteria conferring antibiotic resistance.202336754876
1218170.9911Whole genome sequencing snapshot of multi-drug resistant Klebsiella pneumoniae strains from hospitals and receiving wastewater treatment plants in Southern Romania. We report on the genomic characterization of 47 multi-drug resistant, carbapenem resistant and ESBL-producing K. pneumoniae isolates from the influent (I) and effluent (E) of three wastewater treatment plants (WWTPs) and from Romanian hospital units which are discharging the wastewater in the sampled WWTPs. The K. pneumoniae whole genome sequences were analyzed for antibiotic resistance genes (ARGs), virulence genes and sequence types (STs) in order to compare their distribution in C, I and E samples. Both clinical and environmental samples harbored prevalent and widely distributed ESBL genes, i.e. blaSHV, blaOXA, blaTEM and blaCTX M. The most prevalent carbapenemase genes were blaNDM-1, blaOXA-48 and blaKPC-2. They were found in all types of isolates, while blaOXA-162, a rare blaOXA-48 variant, was found exclusively in water samples. A higher diversity of carbapenemases genes was seen in wastewater isolates. The aminoglycoside modifying enzymes (AME) genes found in all types of samples were aac(6'), ant(2'')Ia, aph(3'), aaD, aac(3) and aph(6). Quinolone resistance gene qnrS1 and the multi-drug resistance oqxA/B pump gene were found in all samples, while qnrD and qnrB were associated to aquatic isolates. The antiseptics resistance gene qacEdelta1 was found in all samples, while qacE was detected exclusively in the clinical ones. Trimethroprim-sulfamethoxazole (dfrA, sul1 and sul2), tetracyclines (tetA and tetD) and fosfomycin (fosA6, known to be located on a transpozon) resistance genes were found in all samples, while for choramphenicol and macrolides some ARGs were detected in all samples (catA1 and catB3 / mphA), while other (catA2, cmIA5 and aac(6')Ib / mphE and msrE) only in wastewater samples. The rifampin resistance genes arr2 and 3 (both carried by class I integrons) were detected only in water samples. The highly prevalent ARGs preferentially associating with aquatic versus clinical samples could ascribe potential markers for the aquatic (blaSHV-145, qacEdelta1, sul1, aadA1, aadA2) and clinical (blaOXA-1, blaSHV-106,blaTEM-150, aac(3)Iia, dfrA14, oqxA10; oqxB17,catB3, tetD) reservoirs of AR. Moreover, some ARGs (oqxA10; blaSHV-145; blaSHV-100, aac(6')Il, aph(3')VI, armA, arr2, cmlA5, blaCMY-4, mphE, msrE, oqxB13, blaOXA-10) showing decreased prevalence in influent versus effluent wastewater samples could be used as markers for the efficiency of the WWTPs in eliminating AR bacteria and ARGs. The highest number of virulence genes (75) was recorded for the I samples, while for E and C samples it was reduced to half. The most prevalent belong to three functional groups: adherence (fim genes), iron acquisition (ent, fep, fyu, irp and ybt genes) and the secretion system (omp genes). However, none of the genes associated with hypervirulent K. pneumoniae have been found. A total of 14 STs were identified. The most prevalent clones were ST101, ST219 in clinical samples and ST258, ST395 in aquatic isolates. These STs were also the most frequently associated with integrons. ST45 and ST485 were exclusively associated with I samples, ST11, ST35, ST364 with E and ST1564 with C samples. The less frequent ST17 and ST307 aquatic isolates harbored blaOXA-162, which was co-expressed in our strains with blaCTX-M-15 and blaOXA-1.202031999747
1242180.9911An Update on Wastewater Multi-Resistant Bacteria: Identification of Clinical Pathogens Such as Escherichia coli O25b:H4-B2-ST131-Producing CTX-M-15 ESBL and KPC-3 Carbapenemase-Producing Klebsiella oxytoca. Wastewater treatment plants (WWTPs) are significant reservoirs of bacterial resistance. This work aims to identify the determinants of resistance produced by Gram-negative bacteria in the influent and effluent of two WWTPs in Portugal. A total of 96 wastewater samples were obtained between 2016 and 2019. The numbers of total aerobic and fecal contamination bacteria were evaluated, and genomic features were searched by polymerase chain reaction (PCR) and Next-Generation Sequencing (NGS). Enterobacteriaceae corresponded to 78.6% (n = 161) of the 205 isolates identified by 16sRNA. The most frequent isolates were Escherichia spp. (57.1%, n = 117), followed by Aeromonas spp. (16.1%, n = 33) and Klebsiella spp. (12.7%, n = 26). The remaining 29 isolates (14.1%) were distributed across 10 different genera. Among the 183 resistant genes detected, 54 isolates produced extended spectrum β-lactamases (ESBL), of which bla(CTX-M-15) was predominant (37 isolates; 68.5%). A KPC-3 carbapenemase-producing K. oxytoca was identified (n = 1), with bla(KPC-3) included in a transposon Tn4401 isoform b. A higher number of virulence genes (VG) (19 genes) was found in the E. coli 5301 (O25b-ST131-B2) isolate compared with a commensal E. coli 5281 (O25b-ST410-A) (six genes). Both shared five VG [Enterobactin; Aerobactin, CFA/1 (clade α); Type1 (clade γ1); Type IV]. In conclusion, this work highlights the role of relevant clinical bacteria in WWTPs, such as KPC-3-producing K. oxytoca, and, for the first time, a CTX-M-15-producing Ochromobactrum intermedium, a human opportunistic pathogen, and a SED-1-producing Citrobacter farmeri, an uncommon CTX-M-type extended-spectrum beta-lactamase.202133799747
1075190.9911Extended Spectrum Beta-Lactamase-Producing Gram-Negative Bacteria Recovered From an Amazonian Lake Near the City of Belém, Brazil. Aquatic systems have been described as antibiotic resistance reservoirs, where water may act as a vehicle for the spread of resistant bacteria and resistance genes. We evaluated the occurrence and diversity of third generation cephalosporin-resistant gram-negative bacteria in a lake in the Amazonia region. This water is used for human activities, including consumption after appropriate treatment. Eighteen samples were obtained from six sites in October 2014. Water quality parameters were generally within the legislation limits. Thirty-three bacterial isolates were identified as Escherichia (n = 7 isolates), Acinetobacter, Enterobacter, and Klebsiella (n = 5 each), Pseudomonas (n = 4), Shigella (n = 3), and Chromobacterium, Citrobacter, Leclercia, Phytobacter (1 isolate each). Twenty nine out of 33 isolates (88%) were resistant to most beta-lactams, except carbapenems, and 88% (n = 29) were resistant to antibiotics included in at least three different classes. Among the beta-lactamase genes inspected, the bla (CTX-M) was the most prevalent (n = 12 positive isolates), followed by bla (TEM) (n = 5) and bla (SHV) (n = 4). bla (CTX-M-15) (n = 5), bla (CTX-M-14) (n = 1) and bla (CTX-M-2) (n = 1) variants were detected in conserved genomic contexts: bla (CTX-M-15) flanked by ISEcp1 and Orf477; bla (CTX-M-14) flanked by ISEcp1 and IS903; and bla (CTX-M-2) associated to an ISCR element. For 4 strains the transfer of bla (CTX-M) was confirmed by conjugation assays. Compared with the recipient, the transconjugants showed more than 500-fold increases in the MICs of cefotaxime and 16 to 32-fold increases in the MICs of ceftazidime. Two isolates (Escherichia coli APC43A and Acinetobacter baumannii APC25) were selected for whole genome analysis. APC43A was predicted as a E. coli pathogen of the high-risk clone ST471 and serotype O154:H18. bla (CTX-M-15) as well as determinants related to efflux of antibiotics, were noted in APC43A genome. A. baumannii APC25 was susceptible to carbapenems and antibiotic resistance genes detected in its genome were intrinsic determinants (e.g., bla (OXA-208) and bla (ADC-like)). The strain was not predicted as a human pathogen and belongs to a new sequence type. Operons related to metal resistance were predicted in both genomes as well as pathogenicity and resistance islands. Results suggest a high dissemination of ESBL-producing bacteria in Lake Água Preta which, although not presenting characteristics of a strongly impacted environment, contains multi-drug resistant pathogenic strains.201930873145