Resistance to aztreonam-avibactam among clinical isolates of E. coli is primarily mediated by altered penicillin binding protein 3 and impermeability.

Herein, we investigated decreased susceptibility (DS; MICs 0.25-4 mg/L) and resistance (R; MICs >4 mg/L) to aztreonam-avibactam (ATM-AVI). Contemporary non-replicate clinical isolates of carbapenemase-producing Escherichia coli (n=90) (CP-EC) and ESBL-producing E. coli (n=12) (EP-EC) was used. CP-EC belonged to 25 distinct sequence types (STs) and all EP-EC belonged to ST405. All strains were isolated through 2019-2022 at the Karolinska University Laboratory, Stockholm, Sweden. ATM-AVI MICs were determined with broth microdilution and the EUCAST epidemiological cutoff value of 0.125 mg/L was used to define the wildtype (WT). Whole genome sequences (Illumina) were analyzed for detecting of resistance determinants among WT vs non-WT isolates. Among 102 isolates, 40 (39%) and 62 (61%) were WT and non-WT respectively. Among non-WT isolates 20 were R and 42 were DS. Resistance was observed among 14/47 NDM-producers, 5/43 OXA-48 group producers, and 1/12 EP-EC. DS was observed among 29/47 NDM, 13/43 OXA-48 group, and 3/12 EP-EC. Resistant isolates predominantly belonged to ST405 followed by STs 410, 361, 167, 617, and 1284. Presence of PBP3 inserts (YRIK/YRIN) were observed in 20/20 and presence of CMY-42 in 5/20 resistant isolates. Several mutations in the ftsI (encoding PBP3) and regulatory genes of outer membrane proteins (OmpC and OmpF) and efflux pumps (AcrAB-TolC) were detected. A ≥2-fold reduction in MICs were observed among 20/20 vs 7/20 isolates tested in the presence of the membrane permeabilizer PMBN and efflux inhibitor PAßN, respectively. In conclusion, resistance to ATM-AVI is a result of interplay of various determinants, including target alterations, deactivating enzymes, and decreased permeability.

Activity of aztreonam/avibactam and ceftazidime/avibactam against Enterobacterales with carbapenemase-independent carbapenem resistance.

Enterobacterales with carbapenemase-independent resistance to carbapenems are sometimes selected during therapy and, on rare occasions, cause outbreaks. Most have extended-spectrum or AmpC ß-lactamases, together with changes to permeability or penicillin-binding proteins (PBPs). Newer ß-lactam-ß-lactamase inhibitor combinations may present useful options for infections due to these organisms. Accordingly, Clinical and Laboratory Standards Institute/European Committee on Antimicrobial Susceptibility Testing broth-microdilution was used to measure the minimum inhibitory concentrations (MICs) of ceftazidime/avibactam and aztreonam/avibactam for 51 carbapenemase-negative Enterobacterales with resistance or reduced susceptibility to carbapenems: genomic sequencing of the least-susceptible organisms was also undertaken. MICs of the two avibactam combinations cross-correlated closely, but with fewer MICs (2/51 vs. 10/51) exceeding 8+4 mg/L in the case of ceftazidime/avibactam. Raised MICs for Escherichia coli were associated with PBP3 inserts together with CMY-42 ß-lactamase; correlates among Enterobacter cloacae complex isolates remain elusive, with AmpC and PBP3 sequences found to be species specific. In the case of Klebsiella spp., no MICs exceeding 2 mg/L were seen for either combination. It appears that these avibactam combinations have potential against Enterobacterales with carbapenemase-independent carbapenem resistance or reduced susceptibility, with ceftazidime/avibactam being more reliably active than aztreonam/avibactam.

Draft genome sequence of NDM-5, CMY-42 carrying carbapenem-resistant Escherichia coli.

We report the draft genome sequence of carbapenem-resistant Escherichia coli (CREC-1) of sequence type (ST2083) isolated from a urine sample of a 2-year-old female toddler carrying antimicrobial resistance genes blaNDM-5 and blaCMY-42 which displays resistance against multiple classes of antibiotics notably ß-lactam antibiotics, cephalosporins, and carbapenem and codes for several virulence factors.

Genetic Features Leading to Reduced Susceptibility to Aztreonam-Avibactam among Metallo-ß-Lactamase-Producing Escherichia coli Isolates.

Metallo-ß-lactamase (MBL)-producing Escherichia coli isolates resistant to the newly developed ß-lactam/ß-lactamase inhibitor drug combination aztreonam-avibactam (ATM-AVI) have been reported. Here, we analyzed a series of 118 clinical MBL-producing E. coli isolates of various geographical origins for susceptibility to ATM-AVI. The nature of the PBP3 protein sequence and the occurrence of blaCMY genes for susceptibility to ATM-AVI were investigated. We showed here that elevated MICs of ATM-AVI among MBL-producing E. coli isolates resulted from a combination of different features, including modification of PBP3 protein sequence through specific amino acid insertions and production of CMY-type enzymes, particularly, CMY-42. We showed here that those insertions identified in the PBP3 sequence are not considered the unique basis of resistance to ATM-AVI, but they significantly contribute to it.

Characterization of O25b-ST131 Escherichia coli Clone Producing CTX-M-15, DHA-4, and CMY-42 in Urinary Tract Infections in a Tunisian Island.

The dissemination of extended-spectrum ß-lactamases encoding genes in Escherichia coli, especially in the uropathogenic O25b-ST131 E. coli clone, constitutes a real concern. We aimed to identify the molecular mechanisms of resistance to cephalosporins among E. coli clinical isolates and to estimate the prevalence of the uropathogenic O25b-ST131 clone in our study. Forty-two cephalosporin-resistant E. coli implicated in urinary tract infections were collected from the Regional Hospital of a southeastern Tunisian Island from April 2015 to August 2016. Molecular screening of ß-lactamases encoding genes by PCR and sequencing showed that the majority of our isolates harbored blaCTX-M gene (blaCTX-M-15 [n = 36], blaCTX-M-14 [n = 2]). Nevertheless, the blaSHV, blaTEM, and blaOXA-1 genes were not detected. Various class C ß-lactamases encoding genes were observed in association or not with blaCTX-M genes and were as follows: blaampC (n = 14), blaCMY-42 (n = 7), blaCMY-2 (n = 1), and blaDHA-4 (n = 1). The research of O25b-ST131 clone was carried out by duplex PCR (pabB and trpA genes) and revealed that most of our isolates (n = 30) belonged to this clone. We also noted that the majority of our isolates belonged to the B2 phylogenetic group (n = 32), five isolates to the B1 phylogenetic group, three isolates to the D phylogenetic group, and only two isolates belonged to the A phylogenetic group. Our study provides new epidemiological information about E. coli clinical isolates in this area. Indeed, this is the first report of CTX-M-14 producing O25b-ST131 E. coli in our country and the first report of DHA-4 and CMY-42 producing E. coli in Tunisia.

Complete Nucleotide Sequence of Plasmids of Two Escherichia coli Strains Carrying bla NDM- 5 and bla NDM - 5 and bla OXA - 181 From the Same Patient.

Aim of this study was to genetically characterize two carbapenemase-producing Escherichia coli strains obtained from a pediatric patient affected by diarrhea, expressing OXA-181 and/or NDM-5 type enzymes. The above microorganisms were collected in the same Desenzano hospital (Northern Italy) where the bla NDM- 5 gene was detected for the first time in Italy 3 years ago. One strain (5P), belonged to sequence type ST405/ST477 (according to Pasture/Oxford schemes) and serotype O102:H6. It was characterized by a 130562 bp multi-replicon plasmid IncFII/IncFIA/IncFIB (pVSI_NDM-5) enclosing two main antibiotic resistance islands: (i) ARI-I, 10030 bp in size, carried genes coding for ß-lactam- (bla OXA- 1, bla CTX-M- 15), fluoroquinolone/aminoglycoside- (aac(6')-lb-cr) and phenicol- resistance (catB3), (ii) ARI-II, 15326 bp in size, carried genes coding for sulfonamide- (sul1), ß-lactam- (bla NDM- 5, bla TEM- 1 B), phenicol- (catB3), trimethoprim- (dfrA17), antiseptic- (qacEΔ1), and aminoglycoside- (aadA5, rmtB) resistance. The other isolate (5M), belonged to sequence type ST2659/ST759 and serotype O50/02:H18, and carried four plasmids: a 153866 bp multi-replicon IncFII/IncFIA/IncFIB (pISV_IncFII_NDM-5), an 89866 bp IncI1 plasmid, a 51480 bp IncX3 plasmid (pISV_IncX3_OXA181), and a 41143 bp IncI plasmid (pISV_IncI_CMY-42). pISV_IncFII_NDM-5 carried two main antibiotic resistance islands: (i) ARI-III, 12220 bp in size, carried genes coding for ß-lactam- (bla OXA- 1), fluoroquinolone/aminoglycoside- (aac(6')-lb-cr), tetracycline- (tet(B)) and phenicol- resistance (catB3, catA1), and ii) ARI-IV, 26527 bp in size, carried determinants coding for macrolide- (erm(B), mph(A)), sulfonamide- (sul1), beta-lactam- (bla NDM- 5, bla TEM- 1 B), trimethoprim- (dfrA14, dfrA12), antiseptic- (qacEΔ1), and aminoglycoside- resistance (aadA5). pISV_IncI_CMY-42 harbored the bla CMY- 42 gene coding for beta-lactam resistance, pISV_IncX3_OXA181 harbored genes encoding fluoroquinolone- (qnrS1) and beta-lactams- resistance (bla OXA- 181). In conclusion, the detection of two different NDM-5 E. coli strains from a pediatric patient with a history of travel to the Far East countries strongly highlight an increasing trend and risk of importation from such areas.

Draft genome sequence of an Escherichia coli ST410 isolate co-harbouring blaCTX-M-15, blaCMY-42, blaOXA-1, aac(3)-IIa and aac(6')-Ib-cr genes with gyrA and parC mutations isolated from a paediatric patient in Poland.

OBJECTIVES: Escherichia coli is one of the major causative agents of nosocomial infections. Here we report the first draft genome sequence of an E. coli strain (no. 158) isolated in Poland carrying blaCTX-M-15, blaCMY-42, blaOXA-1, aac(3)-IIa and aac(6')-Ib-cr genes together with mutations in the gyrA and parC genes. METHODS: Total DNA was sequenced using an Illumina NextSeq 500 platform. The draft genome of E. coli strain 158 was assembled using SPAdes 3.9 assembler. Contigs were annotated using the Prokka v.1.12 algorithm. Species confirmation, multilocus sequence typing (MLST), serotyping, molecular virulence and resistance traits, and plasmid replicons were analysed using appropriate bioinformatics tools available at the Centre for Genomic Epidemiology website. Additional in silico analyses were also conducted. RESULT: The genome size was estimated at 4883487bp, with 4601 predicted coding sequences. The presence of blaCTX-M-15, blaCMY-42, blaOXA-1, aac(3)-IIa and aac(6')-Ib-cr genes was detected in addition to other antimicrobial resistance genes as well as mutations in the gyrA (Ser83Leu and Asp87Asn) and parC (Ser80Ile) genes. The investigated strain E. coli 158 belongs to ST410. CONCLUSION: To our knowledge, this is the first draft genome of an E. coli strain co-harbouring blaCTX-M-15, blaCMY-42, blaOXA-1, aac(3)-IIa and aac(6')-Ib-cr genes with mutations in gyrA and parC reported in Poland. The reported genome sequence contains valuable information on genetic features of antimicrobial resistance mechanisms of E. coli in Poland.

Detection of ST1702 Escherichia coli blaNDM-5 and blaCMY-42 genes positive isolates from a Northern Italian hospital.

We describe two multi drug-resistant (MDR) carbapenemase-producing Escherichia coli clinical isolates from an acute hospital in Milan. Both strains, isolated from a surgical wound sample and a surveillance rectal swab respectively, were positive for a blaNDM-type gene by Xpert Carba-R test. The whole-genome sequence characterization disclosed several resistance determinants: blaNDM-5, blaCMY-42, blaTEM-198, rmtB, mphA. The two isolates belonged to phylogenetic group A, sequence type (ST) 1702 and serotype O89:H9. PCR-based replicon typing and conjugation assay demonstrated an IncI1 plasmid localization for both blaNDM-5 and blaCMY-42 genes. This is the first report of a ST1702 NDM-5 and CMY-42- producing E. coli clone in Italy.

First Report of SHV-148-Type ESBL and CMY-42-Type AmpC ß-Lactamase in Klebsiella pneumoniae Clinical Isolates in Tunisia.

Extended-spectrum beta-lactamase producing Enterobacteriaceae present a real problem worldwide. We aimed to investigate the molecular mechanisms of resistance to antibiotics among Klebsiella pneumoniae clinical isolates collected from a Hospital in the southeast of Tunisia. Eighteen cephalosporin-resistant K. pneumoniae were recovered between April 2015 and August 2016. Molecular characterization of antimicrobial resistance encoding genes was performed by PCR and sequencing. Results revealed several types of Ambler class A ß-lactamase encoding genes among our isolates: [blaCTXM-15 (15), blaSHV-28 (6), blaSHV-1 (2), blaSHV-148 (1), blaSHV-61 (1), blaSHV-76 (1), blaSHV-186 (1), blaTEM-1 (8)]. The association of blaOXA-1 was observed in nine isolates. However, the class C ß-lactamase encoding genes were detected in four isolates [blaCMY-4 (2), blaCMY-42 (1), blaACT-35 (1)]. Molecular typing of K. pneumoniae isolates by pulsed-field gel electrophoresis showed 16 unrelated pulsotypes proving a high diversity among our isolates. Our study provides new epidemiological information showing a huge diversity of ß-lactamase encoding genes among our isolates. In fact, this is the first report of SHV-76, SHV-148, and SHV-186 in Tunisia. This is also the first report of CMY-42 and ACT-35 producing K. pneumoniae in our country.

Occurrence of blaCMY-42 on an IncI1 plasmid in multidrug-resistant Escherichia coli from a tertiary referral hospital in India.

OBJECTIVES: Plasmids of different replicon types are believed to be associated with the carriage and transmission of antimicrobial resistance genes. The present study was undertaken to examine the association of blaCIT with particular plasmid types and to identify Escherichia coli strains involve in the maintenance of this resistance determinant in the plasmid. METHODS: Phenotypic screening of AmpC ß-lactamases was performed by the modified three-dimensional extract method, followed by antimicrobial susceptibility testing and determination of minimum inhibitory concentrations (MICs). Genotyping screening of ß-lactamase genes was performed by PCR assay, followed by sequencing. Transferability of the blaCMY gene was performed by transformation and conjugation experiments. Plasmid incompatibility typing and DNA fingerprinting by enterobacterial repetitive intergenic consensus (ERIC)-PCR were performed. RESULTS: Among 203 E. coli obtained from different clinical specimens (pus, urine, stool and sputum), 37 were detected as harbouring the blaCIT gene and sequencing of this gene showed nucleotide sequence similarity with the blaCMY-42 variant. This study revealed IncI1-type plasmids as carriers of blaCMY-42 and its propagation within E. coli ST5377, ST361 and ST672. According to the stability results, the blaCMY-42-encoding plasmid can be maintained in E. coli strains for a longer duration without any antimicrobial pressure. CONCLUSIONS: These finding document blaCMY-42 on IncI1-type plasmids, which are considered to be the main vehicles for the spread of blaCMY-42 in this hospital setting. Thus, a proper strategy should be developed to curb the expansion of IncI1-type plasmids in the hospital and community environment.

Quinolone co-resistance in ESBL- or AmpC-producing Escherichia coli from an Indian urban aquatic environment and their public health implications.

Quinolone and ß-lactam antibiotics constitute major mainstay of treatment against infections caused by pathogenic Escherichia coli. Presence of E. coli strains expressing co-resistance to both these antibiotic classes in urban aquatic environments which are consistently being used for various anthropogenic activities represents a serious public health concern. From a heterogeneous collection of 61 E. coli strains isolated from the river Yamuna traversing through the National Capital Territory of Delhi (India), those harboring blaCTX-M-15 (n = 10) or blaCMY-42 (n = 2) were investigated for co-resistance to quinolones and the molecular mechanisms thereof. Resistance was primarily attributed to amino acid substitutions in the quinolone resistance-determining regions (QRDRs) of GyrA (S83L ± D87N) and ParC (S80I ± E84K). One of the E. coli strains, viz., IPE, also carried substitutions in GyrB and ParE at positions Ser492→Asn and Ser458→Ala, respectively. The phenotypically susceptible strains nevertheless carried plasmid-mediated quinolone resistance (PMQR) gene, viz., qnrS, which showed co-transfer to the recipient quinolone-sensitive E. coli J53 along with the genes encoding ß-lactamases and led to increase in minimal inhibitory concentrations of quinolone antibiotics. To the best of our knowledge, this represents first report of molecular characterization of quinolone co-resistance in E. coli harboring genes for ESBLs or AmpC ß-lactamases from a natural aquatic environment of India. The study warrants true appreciation of the potential of urban aquatic environments in the emergence and spread of multi-drug resistance and underscores the need to characterize resistance genetic elements vis-à-vis their public health implications, irrespective of apparent phenotypic resistance.