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179900.9019Characterization of a Tigecycline-Resistant and bla(CTX-M)-Bearing Klebsiella pneumoniae Strain from a Peacock in a Chinese Zoo. In Chinese zoos, there are usually specially designed bird parks, similar to petting zoos, that allow children and adults to interact with diverse birds. However, such behaviors present a risk for the transmission of zoonotic pathogens. Recently, we isolated eight strains of Klebsiella pneumoniae and identified two bla(CTX-M)-positive strains from 110 birds, including parrots, peacocks, and ostriches, using anal or nasal swabs in a bird park of a zoo in China. There, K. pneumoniae LYS105A was obtained from a diseased peacock with chronic respiratory diseases by a nasal swab, which harbored the bla(CTX-M-3) gene and exhibited resistance to amoxicillin, cefotaxime, gentamicin, oxytetracycline, doxycycline, tigecycline, florfenicol, and enrofloxacin. According to an analysis by whole-genome sequencing, K. pneumoniae LYS105A belongs to serotype ST859 (sequence type 859)-K19 (capsular serotype 19) and contains two plasmids, of which pLYS105A-2 can be transferred by electrotransformation and harbors numerous resistance genes such as bla(CTX-M-3), aac(6')-Ib-cr5, and qnrB91. The above-mentioned genes are located in a novel mobile composite transposon, Tn7131, which makes horizontal transfer more flexible. Although no known genes were identified in the chromosome, a significant increase in SoxS upregulated the expression levels of phoPQ, acrEF-tolC, and oqxAB, which contributed to strain LYS105A acquiring resistance to tigecycline (MIC = 4 mg/L) and intermediate resistance to colistin (MIC = 2 mg/L). Altogether, our findings show that bird parks in zoos may act as important vehicles for the spread of multidrug-resistant bacteria from birds to humans and vice versa. IMPORTANCE A multidrug-resistant ST859-K19 K. pneumoniae strain, LYS105A, was obtained from a diseased peacock in a Chinese zoo. In addition, multiple resistance genes such as bla(CTX-M-3), aac(6')-Ib-cr5, and qnrB91 were located in a novel composite transposon, Tn7131, of a mobile plasmid, implying that most of the resistance genes in strain LYS105A can be moved easily via horizontal gene transfer. Meanwhile, an increase in SoxS can further positively regulate the expression of phoPQ, acrEF-tolC, and oqxAB, which is the key factor for strain LYS105A to develop resistance to tigecycline and colistin. Taken together, these findings enrich our understanding of the horizontal cross-species spread of drug resistance genes, which will help us curb the development of bacterial resistance.202336809063
138910.8913Whole-Genome Sequencing of Gram-Negative Bacteria Isolated From Bovine Mastitis and Raw Milk: The First Emergence of Colistin mcr-10 and Fosfomycin fosA5 Resistance Genes in Klebsiella pneumoniae in Middle East. Antimicrobial resistance is a major concern in the dairy industry. This study investigated the prevalence, antimicrobial resistance phenotypes, and genome sequencing of Gram-negative bacteria isolated from clinical (n = 350) and subclinical (n = 95) bovine mastitis, and raw unpasteurized milk (n = 125). Klebsiella pneumoniae, Aeromonas hydrophila, Enterobacter cloacae (100% each), Escherichia coli (87.78%), and Proteus mirabilis (69.7%) were the most prevalent multidrug-resistant (MDR) species. Extensive drug-resistance (XDR) phenotype was found in P. mirabilis (30.30%) and E. coli (3.33%) isolates. Ten isolates (four E. coli, three Klebsiella species and three P. mirabilis) that displayed the highest multiple antibiotic resistance (MAR) indices (0.54-0.83), were exposed to whole-genome sequencing (WGS). Two multilocus sequence types (MLST): ST2165 and ST7624 were identified among the sequenced E. coli isolates. Three E. coli isolates (two from clinical mastitis and one from raw milk) belonging to ST2165 showed similar profile of plasmid replicon types: IncFIA, IncFIB, IncFII, and IncQ1 with an exception to an isolate that contained IncR, whereas E. coli ST7624 showed a different plasmid profile including IncHI2, IncHI2A, IncI1α, and IncFII replicon types. ResFinder findings revealed the presence of plasmid-mediated colistin mcr-10 and fosfomycin fosA5 resistance genes in a K. pneumoniae (K1) isolate from bovine milk. Sequence analysis of the reconstructed mcr-10 plasmid from WGS of K1 isolate, showed that mcr-10 gene was bracketed by xerC and insertion sequence IS26 on an IncFIB plasmid. Phylogenetic analysis revealed that K1 isolate existed in a clade including mcr-10-harboring isolates from human and environment with different STs and countries [United Kingdom (ST788), Australia (ST323), Malawi (ST2144), Myanmar (ST705), and Laos (ST2355)]. This study reports the first emergence of K. pneumoniae co-harboring mcr-10 and fosA5 genes from bovine milk in the Middle East, which constitutes a public health threat and heralds the penetration of the last-resort antibiotics. Hence, prudent use of antibiotics in both humans and animals and antimicrobial surveillance plans are urgently required.202134956131
138820.8909Snapshot Study of Whole Genome Sequences of Escherichia coli from Healthy Companion Animals, Livestock, Wildlife, Humans and Food in Italy. Animals, humans and food are all interconnected sources of antimicrobial resistance (AMR), allowing extensive and rapid exchange of AMR bacteria and genes. Whole genome sequencing (WGS) was used to characterize 279 Escherichia coli isolates obtained from animals (livestock, companion animals, wildlife), food and humans in Italy. E. coli predominantly belonged to commensal phylogroups B1 (46.6%) and A (29%) using the original Clermont criteria. One hundred and thirty-six sequence types (STs) were observed, including different pandemic (ST69, ST95, ST131) and emerging (ST10, ST23, ST58, ST117, ST405, ST648) extraintestinal pathogenic Escherichia coli (ExPEC) lineages. Eight antimicrobial resistance genes (ARGs) and five chromosomal mutations conferring resistance to highest priority critically important antimicrobials (HP-CIAs) were identified (qnrS1, qnrB19, mcr-1, bla(CTX-M1,15,55), bla(CMY-2), gyrA/parC/parE, ampC and pmrB). Twenty-two class 1 integron arrangements in 34 strains were characterized and 11 ARGs were designated as intI1 related gene cassettes (aadA1, aadA2, aadA5, aad23, ant2_Ia, dfrA1, dfrA7, dfrA14, dfrA12, dfrA17, cmlA1). Notably, most intI1 positive strains belonged to rabbit (38%) and poultry (24%) sources. Three rabbit samples carried the mcr-1 colistin resistance gene in association with IS6 family insertion elements. Poultry meat harbored some of the most prominent ExPEC STs, including ST131, ST69, ST10, ST23, and ST117. Wildlife showed a high average number of virulence-associated genes (VAGs) (mean = 10), mostly associated with an ExPEC pathotype and some predominant ExPEC lineages (ST23, ST117, ST648) were identified.202033172096
520530.8909Antimicrobial resistance and virulence factors of Klebsiella quasipneumoniae, the novel sequence types (ST) 7979 and 7980 from Indonesia. Klebsiella pneumoniae is a human pathogen of global concern. The more recently described pathogen, K. quasipneumoniae, shares similar morphological characteristics with K. pneumoniae and is commonly misidentified as this species using conventional laboratory techniques. This study investigates the molecular characteristics of four phenotype-identified K. pneumoniae isolates obtained from hospital wastewater in Jakarta, Indonesia. Whole-genome sequencing (WGS) and the Average Nucleotide Identity (ANI) showed that these isolates were eventually identified as K. quasipneumoniae subsp. quasipneumoniae, a closely related species of K. pneumoniae. These isolates of novel ST7979 and ST7980 strains are classified as multi-drug resistant (MDR) bacteria and harbor many antibiotic-resistance genes. Interestingly, the novel ST7980 strain is carbapenem non-susceptible and harbors the sul1 gene and the heat-stable enterotoxin gene, astA. The ST7979 strains have KL55 capsular type and O3b type, whereas the ST7980 strains have KL107 and O12 types. Our finding highlights the significance of identifying the K. quasipneumoniae strain utilizing a genomic platform. Additionally, routine surveillance is needed to monitor the hospital wastewater and avoid the spread of multidrug-resistant bacteria.202540609771
82740.8908Characterization of a ST137 multidrug-resistant Campylobacter jejuni strain with a tet(O)-positive genomic island from a bloodstream infection patient. Campylobacter jejuni (C. jejuni) is a major cause of gastroenteritis and rarely cause bloodstream infection. Herein, we characterized a multidrug-resistant C. jejuni strain LZCJ isolated from a tumor patient with bloodstream infection. LZCJ was resistant to norfloxacin, ampicillin, ceftriaxone, ciprofloxacin and tetracycline. It showed high survival rate in serum and acidic environment. Whole genome sequencing (WGS) analysis revealed that strain LZCJ had a single chromosome of 1,629,078 bp (30.6 % G + C content) and belonged to the ST137 lineage. LZCJ shared the highest identity of 99.66 % with the chicken-derived C. jejuni MTVDSCj20. Four antimicrobial resistance genes (ARGs) were detected, bla(OXA-61), tet(O), gyrA (T86I), and cmeR (G144D and S207G). In addition, a 12,746 bp genomic island GI_LZCJ carrying 15 open reading frames (ORFs) including the resistance gene tet(O) was identified. Sequence analysis found that the GI_LZCJ was highly similar to the duck-derived C. jejuni ZS004, but with an additional ISChh1-like sequence. 137 non-synonymous mutations in motility related genes (flgF, fapR, flgS), capsular polysaccharide (CPS) coding genes (kpsE, kpsF, kpsM, kpsT), metabolism associated genes (nuoF, nuoG, epsJ, holB), and transporter related genes (comEA, gene0911) were confirmed in LZCJ compared with the best closed chicken-derived strain MTVDSCj20. Our study showed that C. jejuni strain LZCJ was highly similar to the chicken-derived strain MTVDSCj20 but with a lot of SNPs involved in motility, CPS and metabolism coding genes. This strain possessed a tet(O)-positive genomic island GI_LZCJ, which was closed to duck-derived C. jejuni ZS004, but with an additional ISChh1-like sequence. The above data indicated that the LZCJ strain may originate from foodborne bacteria on animals and the importance of continuous surveillance for the spread of foodborne bacteria.202439208964
144150.8907Molecular characterisation of carbapenem-resistant Klebsiella pneumoniae clinical isolates: preliminary experience from a tertiary care teaching hospital in the Himalayas. BACKGROUND: There is a lack of whole-genome sequencing (WGS) data on multidrug-resistant (MDR) bacteria from the Uttarakhand region of India. The aim of this study was to generate WGS data of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates recovered from patients in Uttarakhand's tertiary care centre. METHODS: A cross-sectional study included 29 MDR K. pneumoniae test isolates obtained from various clinical samples submitted to the bacteriology laboratory for culture and sensitivity testing from July 2018 to August 2019. After preliminary identification and antibiotic susceptibility testing, these isolates were subjected to WGS. RESULTS: A total of 27 of 29 isolates were CRKP. ST14 was the most common sequence type (n=8 [29.6%]). Carbapenem resistance was mainly encoded by OXA-48-like genes (21/27 [77.8%]). All isolates had a varied arsenal of resistance genes to different antibiotic classes. KL2 (9/27 [33.3%]) and KL51 (8/27 [29.6%]) were dominant K loci types. O1 and O2 together accounted for 88.9% (n=27) of CRKP isolates. Genes encoding yersiniabactin (ybt) and aerobactin (iuc) were identified in 88.9% (24/27) and 29.6% (8/27) of isolates. The predominant plasmid replicons present were ColKP3 (55.5%), IncFII(K) (51.8%) and IncFIB(pQil) (44.4%). CONCLUSIONS: This study emphasises the need for continued genomic surveillance of MDR bacteria that could be instrumental in developing treatment guidelines based on integrating phenotypic and molecular methods.202235029688
140960.8905Detection of diverse carbapenem and multidrug resistance genes and high-risk strain types among carbapenem non-susceptible clinical isolates of target gram-negative bacteria in Kenya. Carbapenem-resistant gram-negative bacteria are an increasingly significant clinical threat globally. This risk may be underestimated in Kenya as only four carbapenemase genes in three bacterial species have been described. The study aimed to understand the antibiotic resistance profiles, genes, sequence types, and distribution of carbapenem-resistant gram-negative bacteria from patients in six hospitals across five Kenyan counties by bacterial culture, antibiotic susceptibility testing, and whole-genome sequence analysis. Forty-eight, non-duplicate, carbapenem non-susceptible, clinical isolates were identified across the five counties (predominantly in Nairobi and Kisii): twenty-seven Acinetobacter baumannii, fourteen Pseudomonas aeruginosa, three Escherichia coli, two Enterobacter cloacae, and two Klebsiella pneumoniae. All isolates were non-susceptible to β-lactam drugs with variable susceptibility to tigecycline (66%), minocycline (52.9%), tetracycline (29.4%), and levofloxacin (22.9%). Thirteen P. aeruginosa isolates were resistant to all antibiotics tested. Eleven carbapenemase genes were identified: blaNDM-1, blaOXA-23, -58, -66, -69, and -91 in A. baumannii (STs 1, 2, 164 and a novel ST1475), blaNDM-1 in E. cloacae (STs 25,182), blaNDM-1, blaVIM-1and -6, blaOXA-50 in P. aeruginosa (STs 316, 357, 654, and1203), blaOXA-181, blaNDM-1 in K. pneumoniae (STs 147 and 219), and blaNDM-5 in E. coli (ST164). Five A. baumannii isolates had two carbapenemases, blaNDM-1, and either blaOXA-23 (4) or blaOXA-58 (1). AmpC genes were detected in A. baumannii (blaADC-25), E. cloacae (blaDHA-1 and blaACT-6, 16), and K. pneumoniae (blaCMY). Significant multiple-drug resistant genes were the pan-aminoglycoside resistance16srRNA methyltransferase armA, rmtB, rmtC, and rmtF genes. This study is the first to report blaOXA-420, -58, -181, VIM-6, and blaNDM-5 in Kenyan isolates. High-risk STs of A. baumannii (ST1475, ST2), E. cloacae ST182, K. pneumoniae ST147, P. aeruginosa (ST357, 654), and E. coli ST167, ST648 were identified which present considerable therapeutic danger. The study recommends urgent carbapenem use regulation and containment of high-risk carbapenem-resistant bacteria.202133617559
138670.8904ESBL/pAmpC-producing Enterobacterales in common leopard geckos (Eublepharis macularius) and central bearded dragons (Pogona vitticeps) from Portugal. Common leopard geckos (Eublepharis macularius) and central bearded dragon (Pogona vitticeps) are widely kept as pets but can harbor pathogenic bacteria, including antimicrobial-resistant (AMR) bacteria. This study aimed to research the frequency of β-lactamase-producing Enterobacterales in these two reptile species. A total of 132 samples were collected from the oral and cloacal cavities of healthy common leopard geckos and central bearded dragons in the Lisbon area, Portugal. Antimicrobial resistance was assessed for third-generation cephalosporin (3GC)-resistant Enterobacterales. The results revealed that 3GC-resistant Enterobacterales were observed in 17.9% (n = 14/78) of the reptiles. The most commonly identified species were: Citrobacter freundii and Klebsiella aerogenes. Furthermore, some isolates produced extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases (AmpC) encoding genes such as bla (CMY-2), bla (CTX-M-15,) and bla (TEM-1). These findings emphasize the potential role of these reptiles in the spread of AMR bacteria, particularly in urban settings where human- animal interactions are frequent. Given the zoonotic risks, this study emphasizes the importance of continued surveillance and responsible antimicrobial use in both veterinary and human medicine to mitigate the spread of AMR bacteria.202540370835
138580.8899GENOMIC CHARACTERIZATION OF MULTIDRUG-RESISTANT EXTENDED-SPECTRUM β-LACTAMASE-PRODUCING ESCHERICHIA COLI AND KLEBSIELLA PNEUMONIAE FROM CHIMPANZEES (PAN TROGLODYTES) FROM WILD AND SANCTUARY LOCATIONS IN UGANDA. Farm and wild animals may serve as reservoirs of antimicrobial-resistant bacteria of human health relevance. We investigated the occurrence and genomic characteristics of extended spectrum β-lactamase (ESBL)-producing bacteria in Ugandan chimpanzees (Pan troglodytes) residing in two environments with or without close contact to humans. The ESBL-producing Escherichia coli and Klebsiella pneumoniae were isolated from fecal material of chimpanzees from Budongo Forest and Ngamba Island Chimpanzee Sanctuary in Uganda and were more commonly isolated from chimpanzees in Ngamba Island Chimpanzee Sanctuary, where animals have close contact with humans. Selected ESBL isolates (E. coli n=9, K. pneumoniae n=7) were analyzed by whole-genome sequencing to determine the presence of resistance genes, as well as sequence type and virulence potential; the blaCTX-M-15 gene was present in all strains. Additionally, the ESBL genes blaSHV-11 and blaSHV-12 were found in strains in the study. All strains were found to be multidrug resistant. The E. coli strains belonged to four sequence types (ST2852, ST215, ST405, and ST315) and the K. pneumoniae strains to two sequence types (ST1540 and ST597). Virulence genes did not indicate that strains were of common E. coli pathotype, but strains with the same sequence types as isolated in the current study have previously been reported from clinical cases in Africa. The findings indicate that chimpanzees in close contact with humans may carry ESBL bacteria at higher frequency than those in the wild, indicating a potential anthropogenic transmission.202235255126
84390.8896Whole Genome Sequencing Reveals Presence of High-Risk Global Clones of Klebsiella pneumoniae Harboring Multiple Antibiotic Resistance Genes in Multiple Plasmids in Mwanza, Tanzania. BACKGROUND: Klebsiella pneumoniae is an important multidrug-resistant (MDR) pathogen, causing both community- and healthcare-associated infections. The resistance is due to the continuous accumulation of multiple antibiotic-resistance-genes (ARGs) through spontaneous genomic mutations and the acquisition of conjugative plasmids. This study presents antibiotics resistance genes, plasmids replicons, and virulence genes of K. pneumoniae isolates from clinical specimens in a tertiary hospital, Mwanza, Tanzania. METHODS: Whole genome sequencing (WGS) of 34 K. pneumoniae was performed, using an Illumina NextSeq 500, followed by in silco analysis. RESULTS: A total of 34 extended-spectrum beta-lactamase-producing K. pneumoniae, isolated from blood samples from neonatal units were whole-genome sequenced. Of these, 28 (82.4%) had an identified sequence type (ST), with ST14 (39.3%, n = 11) being frequently identified. Moreover, 18 (52.9%) of the bacteria harbored at least one plasmid, from which a total of 25 plasmid replicons were identified with a predominance of IncFIB(K) 48.0% (n = 12). Out of 34 sequenced K. pneumoniae, 32 (94.1%) were harboring acquired antibiotic/biocides-resistance-genes (ARGs) with a predominance of bla(CTX-M-15) (90.6%), followed by oqxB (87.5%), oqxA (84.4%), bla(TEM-1B) (84.4%) and sul2 (84.4%). Interestingly, we observed the ColRNAI plasmid-replicon (n = 1) and qacE gene (n = 4) for the first time in this setting. CONCLUSION: Global high-risk clones of K. pneumoniae isolates carry multiple ARGs in multiple plasmid-replicons. Findings from this study warrant genomic-based surveillance to monitor high-risk global clones, epidemic plasmids and ARGs in low- and middle-income countries.202236557648
1535100.8895Complete 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
1241110.8895Spectrum of Bacterial Colonization in Patients Hospitalized for Treatment of Multidrug-Resistant Tuberculosis. This study investigated the bacterial colonization in patients admitted for treatment of drug-resistant tuberculosis in a specialized TB hospital. Identification and antimicrobial susceptibility testing of bacterial isolates (n = 62) from nasal, groin, and rectal swabs [patient cohort (n = 37)] were determined by the VITEK-MS system. Resistance gene analysis was by PCR and DNA sequencing. Molecular typing of Klebsiella pneumoniae isolates was by Multilocus Sequencing Typing (MLST). Patients (n = 13/37; 35%) were colonized by multidrug-resistant (MDR) bacteria (ESBL and MRSA) on admission. Of the 24 patients who were not colonized by MDR bacteria on admission, 46% (17/37) became colonized by MDR bacteria within 1 month of admission, mostly with ESBL-producing Enterobacteriales and resistance to aminoglycosides and fluoroquinolones. ESBL Escherichia coli (41/62; 66%) and K. pneumoniae (14/62; 23%) predominated. Genes encoding for ESBLs (bla(CTX-M-14), bla(CTX-M-15), bla(SHV-28), bla(OXA-1), and bla(OXY-2)) and plasmid-mediated quinolone resistant genes (qnrB1, qnrB4, and qnrB10) were detected. MLST revealed genetic diversity among the K. pneumoniae isolates from hospitalized patients. This study provides insight into bacterial pathogen colonization in hospitalized TB patients with the first occurrence of the qnrB4 and qnrB10 genes and co-expression of genes: qnrB4+aac(6')-lb-cr, qnrB10+aac(6')-lb-cr, qnrB4+qnrS1, and qnrB10+qnrS1 in fluoroquinolone-resistant E. coli isolates within South Africa. However, the source and colonization routes of these isolates could not be determined.202133074767
1538120.8892KPC-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
1995130.8891Genomic insights into Shigella species isolated from small ruminants and manure in the North West Province, South Africa. This study investigated Shigella species' antibiotic resistance patterns and genomic characteristics from small ruminants and manure collected in Potchefstroom, North West, South Africa. Whole genome sequencing was used to determine resistome profiles of Shigella flexneri isolates from small ruminants' manure and Shigella boydii from sheep faeces. Comparative genomics was employed on the South African 261 S. flexneri strains available from GenBank, including the sequenced strains in this study, by investigating the serovars, antibiotic resistance genes (ARGs), and plasmid replicon types. The S. flexneri strains could not be assigned to known sequence types, suggesting novel or uncharacterized lineages. S. boydii R7-1A was assigned to sequence type 202 (ST202). Serovar 2A was the most common among South African S. flexneri strains, found in 96% of the 250 compared human-derived isolates. The shared mdf(A) was the most prevalent gene, identified in 99% of 261 S. flexneri genomes, including plasmid replicon types ColRNAI_1 (99%) and IncFII_1 (98%). Both species share a core set of resistance determinants mainly involving β-lactams (ampC1, ampC, ampH), macrolides (mphB), polymyxins (eptA, pmrF), multidrug efflux pumps (AcrAB-TolC, Mdt, Emr, Kpn families), and regulatory systems (marA, hns, crp, baeRS, evgAS, cpxA, gadX). However, S. boydii possesses additional resistance genes conferring resistance to tetracyclines (tet(A)), phenicols (floR), sulphonamides (sul2), and aminoglycosides (APH(3'')-Ib, APH(6)-Id), along with the acrEF efflux pump components (acrE, acrF). In contrast, S. flexneri harboured unique genes linked to polymyxin resistance (ugd) and regulatory functions (sdiA, gadW) that were absent in S. boydii. These findings highlight Shigella strains' genomic diversity and antimicrobial resistance potential in livestock-associated environments. Moreover, S. boydii highlights the potential risk of multidrug-resistant bacteria in farming and environmental routes. KEY POINTS: • First whole genome study of Shigella from manure and small ruminants in South Africa. • Shigella boydii strain carried multiple resistance genes to β-lactams and tetracycline. • Multidrug efflux pump gene mdf(A) was detected in 99% of South African Shigella flexneri strains.202541148367
959140.8890Analyzing Antibiotic Resistance in Bacteria from Wastewater in Pakistan Using Whole-Genome Sequencing. Background: Wastewater is a major source of Antibiotic-Resistant Bacteria (ARB) and a hotspot for the exchange of Antibiotic-Resistant Genes (ARGs). The occurrence of Carbapenem-Resistant Bacteria (CRB) in wastewater samples is a major public health concern. Objectives: This study aimed to analyze Antibiotic resistance in bacteria from wastewater sources in Pakistan. Methods: We analyzed 32 bacterial isolates, including 18 Escherichia coli, 4 Klebsiella pneumoniae, and 10 other bacterial isolates using phenotypic antibiotic susceptibility assay and whole-genome sequencing. This study identified the ARGs, plasmid replicons, and integron genes cassettes in the sequenced isolates. One representative isolate was further sequenced using Illumina and Oxford nanopore sequencing technologies. Results: Our findings revealed high resistance to clinically important antibiotics: 91% of isolates were resistant to cefotaxime, 75% to ciprofloxacin, and 62.5% to imipenem, while 31% showed non-susceptibility to gentamicin. All E. coli isolates were resistant to cephalosporins, with 72% also resistant to carbapenems. Sequence analysis showed a diverse resistome, including carbapenamases (blaNDM-5, blaOXA-181), ESBLs (blaCTX-M-15, blaTEM), and AmpC-type β-lactamases (blaCMY). Key point mutations noticed in the isolates were pmrB_Y358N (colistin) and ftsI_N337NYRIN, ftsI_I336IKYRI (carbapenem). The E. coli isolates had 11 different STs, with ST410 predominating (28%). Notably, the E. coli phylogroup A isolate 45EC1, (ST10886) is reported for the first time from wastewater, carrying blaNDM-5, blaCMY-16, and pmrB_Y358N with class 1 integron gene cassette of dfrA12-aadA2-qacEΔ1 on a plasmid-borne contig. Other carbapenamase, blaNDM-1 and blaOXA-72, were detected in K. pneumoniae 22EB1 and Acinetobacter baumannii 51AC1, respectively. The integrons with the gene cassettes encoding antibiotic resistance, and the transport and bacterial mobilization protein, were identified in the sequenced isolates. Ten plasmid replicons were identified, with IncFIB prevalent in 53% of isolates. Combined Illumina and Oxford nanopore sequencing revealed blaNDM-5 on an IncFIA/IncFIC plasmid and is identical to those reported in the USA, Myanmar, and Tanzania. Conclusions: These findings highlight the environmental prevalence of high-risk and WHO-priority pathogens with clinically important ARGs, underscoring the need for a One Health approach to mitigate ARB isolates.202439452204
1387150.8890Whole-Genome Characterisation of ESBL-Producing E. coli Isolated from Drinking Water and Dog Faeces from Rural Andean Households in Peru. E. coli that produce extended-spectrum β-lactamases (ESBLs) are major multidrug-resistant bacteria. In Peru, only a few reports have characterised the whole genome of ESBL enterobacteria. We aimed to confirm the identity and antimicrobial resistance (AMR) profile of two ESBL isolates from dog faeces and drinking water of rural Andean households and determine serotype, phylogroup, sequence type (ST)/clonal complex (CC), pathogenicity, virulence genes, ESBL genes, and their plasmids. To confirm the identity and AMR profiles, we used the VITEK(®)2 system. Whole-genome sequencing (WGS) and bioinformatics analysis were performed subsequently. Both isolates were identified as E. coli, with serotypes -:H46 and O9:H10, phylogroups E and A, and ST/CC 5259/- and 227/10, respectively. The isolates were ESBL-producing, carbapenem-resistant, and not harbouring carbapenemase-encoding genes. Isolate 1143 ST5259 harboured the astA gene, encoding the EAST(1) heat-stable toxin. Both genomes carried ESBL genes (bla(EC-15), bla(CTX-M-8), and bla(CTX-M-55)). Nine plasmids were detected, namely IncR, IncFIC(FII), IncI, IncFIB(AP001918), Col(pHAD28), IncFII, IncFII(pHN7A8), IncI1, and IncFIB(AP001918). Finding these potentially pathogenic bacteria is worrisome given their sources and highlights the importance of One-Health research efforts in remote Andean communities.202235625336
2632160.8889Environmental Spread of Extended Spectrum Beta-Lactamase (ESBL) Producing Escherichia coli and ESBL Genes among Children and Domestic Animals in Ecuador. BACKGROUND: There is a significant gap in our understanding of the sources of multidrug-resistant bacteria and resistance genes in community settings where human-animal interfaces exist. OBJECTIVES: This study characterized the relationship of third-generation cephalosporin-resistant Escherichia coli (3GCR-EC) isolated from animal feces in the environment and child feces based on phenotypic antimicrobial resistance (AMR) and whole genome sequencing (WGS). METHODS: We examined 3GCR-EC isolated from environmental fecal samples of domestic animals and child fecal samples in Ecuador. We analyzed phenotypic and genotypic AMR, as well as clonal relationships (CRs) based on pairwise single-nucleotide polymorphisms (SNPs) analysis of 3GCR-EC core genomes. CRs were defined as isolates with fewer than 100 different SNPs. RESULTS: A total of 264 3GCR-EC isolates from children (n = 21), dogs (n = 20), and chickens (n = 18) living in the same region of Quito, Ecuador, were identified. We detected 16 CRs total, which were found between 7 children and 5 domestic animals (5 CRs) and between 19 domestic animals (11 CRs). We observed that several clonally related 3GCR-EC isolates had acquired different plasmids and AMR genes. Most CRs were observed in different homes (n = 14) at relatively large distances. Isolates from children and domestic animals shared the same blaCTX-M allelic variants, and the most prevalent were blaCTX-M-55 and blaCTX-M-65, which were found in isolates from children, dogs, and chickens. DISCUSSION: This study provides evidence of highly dynamic horizontal transfer of AMR genes and mobile genetic elements (MGEs) in the E. coli community and shows that some 3GCR-EC and (extended-spectrum β-lactamase) ESBL genes may have moved relatively large distances among domestic animals and children in semirural communities near Quito, Ecuador. Child-animal contact and the presence of domestic animal feces in the environment potentially serve as important sources of drug-resistant bacteria and ESBL genes. https://doi.org/10.1289/EHP7729.202133617318
1798170.8888Impacts of Domestication and Veterinary Treatment on Mobile Genetic Elements and Resistance Genes in Equine Fecal Bacteria. Antimicrobial resistance in bacteria is a threat to both human and animal health. We aimed to understand the impact of domestication and antimicrobial treatment on the types and numbers of resistant bacteria, antibiotic resistance genes (ARGs), and class 1 integrons (C1I) in the equine gut microbiome. Antibiotic-resistant fecal bacteria were isolated from wild horses, healthy farm horses, and horses undergoing veterinary treatment, and isolates (9,083 colonies) were screened by PCR for C1I; these were found at frequencies of 9.8% (vet horses), 0.31% (farm horses), and 0.05% (wild horses). A collection of 71 unique C1I(+) isolates (17 Actinobacteria and 54 Proteobacteria) was subjected to resistance profiling and genome sequencing. Farm horses yielded mostly C1I(+) Actinobacteria (Rhodococcus, Micrococcus, Microbacterium, Arthrobacter, Glutamicibacter, Kocuria), while vet horses primarily yielded C1I(+) Proteobacteria (Escherichia, Klebsiella, Enterobacter, Pantoea, Acinetobacter, Leclercia, Ochrobactrum); the vet isolates had more extensive resistance and stronger P(C) promoters in the C1Is. All integrons in Actinobacteria were flanked by copies of IS6100, except in Micrococcus, where a novel IS5 family element (ISMcte1) was implicated in mobilization. In the Proteobacteria, C1Is were predominantly associated with IS26 and also IS1, Tn21, Tn1721, Tn512, and a putative formaldehyde-resistance transposon (Tn7489). Several large C1I-containing plasmid contigs were retrieved; two of these (plasmid types Y and F) also had extensive sets of metal resistance genes, including a novel copper-resistance transposon (Tn7519). Both veterinary treatment and domestication increase the frequency of C1Is in equine gut microflora, and each of these anthropogenic factors selects for a distinct group of integron-containing bacteria. IMPORTANCE There is increasing acknowledgment that a "one health" approach is required to tackle the growing problem of antimicrobial resistance. This requires that the issue is examined from not only the perspective of human medicine but also includes consideration of the roles of antimicrobials in veterinary medicine and agriculture and recognizes the importance of other ecological compartments in the dissemination of ARGs and mobile genetic elements such as C1I. We have shown that domestication and veterinary treatment increase the frequency of occurrence of C1Is in the equine gut microflora and that, in healthy farm horses, the C1I are unexpectedly found in Actinobacteria, while in horses receiving antimicrobial veterinary treatments, a taxonomic shift occurs, and the more typical integron-containing Proteobacteria are found. We identified several new mobile genetic elements (plasmids, insertion sequences [IS], and transposons) on genomic contigs from the integron-containing equine bacteria.202336988354
5206180.8887Draft genome sequence of an extensively drug-resistant Pseudomonas aeruginosa isolate belonging to ST644 isolated from a footpad infection in a Magellanic penguin (Spheniscus magellanicus). OBJECTIVES: The incidence of multidrug-resistant bacteria in wildlife animals has been investigated to improve our knowledge of the spread of clinically relevant antimicrobial resistance genes. The aim of this study was to report the first draft genome sequence of an extensively drug-resistant (XDR) Pseudomonas aeruginosa ST644 isolate recovered from a Magellanic penguin with a footpad infection (bumblefoot) undergoing rehabilitation process. METHODS: The genome was sequenced on an Illumina NextSeq(®) platform using 150-bp paired-end reads. De novo genome assembly was performed using Velvet v.1.2.10, and the whole genome sequence was evaluated using bioinformatics approaches from the Center of Genomic Epidemiology, whereas an in-house method (mapping of raw whole genome sequence reads) was used to identify chromosomal point mutations. RESULTS: The genome size was calculated at 6436450bp, with 6357 protein-coding sequences and the presence of genes conferring resistance to aminoglycosides, β-lactams, phenicols, sulphonamides, tetracyclines, quinolones and fosfomycin; in addition, mutations in the genes gyrA (Thr83Ile), parC (Ser87Leu), phoQ (Arg61His) and pmrB (Tyr345His), conferring resistance to quinolones and polymyxins, respectively, were confirmed. CONCLUSION: This draft genome sequence can provide useful information for comparative genomic analysis regarding the dissemination of clinically significant antibiotic resistance genes and XDR bacterial species at the human-animal interface.201829277728
1992190.8885Antimicrobial Resistance Genes, Cassettes, and Plasmids Present in Salmonella enterica Associated With United States Food Animals. The ability of antimicrobial resistance (AR) to transfer, on mobile genetic elements (MGEs) between bacteria, can cause the rapid establishment of multidrug resistance (MDR) in bacteria from animals, thus creating a foodborne risk to human health. To investigate MDR and its association with plasmids in Salmonella enterica, whole genome sequence (WGS) analysis was performed on 193 S. enterica isolated from sources associated with United States food animals between 1998 and 2011; 119 were resistant to at least one antibiotic tested. Isolates represented 86 serotypes and variants, as well as diverse phenotypic resistance profiles. A total of 923 AR genes and 212 plasmids were identified among the 193 strains. Every isolate contained at least one AR gene. At least one plasmid was detected in 157 isolates. Genes were identified for resistance to aminoglycosides (n = 472), β-lactams (n = 84), tetracyclines (n = 171), sulfonamides (n = 91), phenicols (n = 42), trimethoprim (n = 8), macrolides (n = 5), fosfomycin (n = 48), and rifampicin (n = 2). Plasmid replicon types detected in the isolates were A/C (n = 32), ColE (n = 76), F (n = 43), HI1 (n = 4), HI2 (n = 20), I1 (n = 62), N (n = 4), Q (n = 7), and X (n = 35). Phenotypic resistance correlated with the AR genes identified in 95.4% of cases. Most AR genes were located on plasmids, with many plasmids harboring multiple AR genes. Six antibiotic resistance cassette structures (ARCs) and one pseudo-cassette were identified. ARCs contained between one and five resistance genes (ARC1: sul2, strAB, tetAR; ARC2: aac3-iid; ARC3: aph, sph; ARC4: cmy-2; ARC5: floR; ARC6: tetB; pseudo-ARC: aadA, aac3-VIa, sul1). These ARCs were present in multiple isolates and on plasmids of multiple replicon types. To determine the current distribution and frequency of these ARCs, the public NCBI database was analyzed, including WGS data on isolates collected by the USDA Food Safety and Inspection Service (FSIS) from 2014 to 2018. ARC1, ARC4, and ARC5 were significantly associated with cattle isolates, while ARC6 was significantly associated with chicken isolates. This study revealed that a diverse group of plasmids, carrying AR genes, are responsible for the phenotypic resistance seen in Salmonella isolated from United States food animals. It was also determined that many plasmids carry similar ARCs.201931057528