Molecular insights into the evolutionary trajectory of a Klebsiella aerogenes clinical isolate with a complex trade-off between resistance and virulence.

The fitness cost associated with antimicrobial resistance has an important influence on evolutionary dynamics. We compared the genomes of three Klebsiella aerogenes isolates recovered from blood samples or deep abscess cultures from the same patient: the wild-type strain (CT_WT), a piperacillin-tazobactam-resistant strain (CT_PENI), and an extended-spectrum-cephalosporin (ESC)-resistant strain (CT_R). Whole-genome sequencing revealed that CT_PENI had acquired a TEM-1 ß-lactamase with a mutated promoter, accounting for overproduction. CT_PENI then acquired an E240G substitution in the TEM-1 ß-lactamase (resulting in TEM-207) and lost the porin-encoding ompK36 gene to give CT_R. All three strains showed the same virulence in a mouse model of intraperitoneal infection. The results of recombination and transformation assays indicated that when present separately, the TEM-207 overproduction and the ompK36 gene deletion had only small effects on susceptibility to ESCs. However, the combination of the two changes led to a much lower susceptibility to ESCs. Moreover, the levels of fitness in vitro and in vivo in a murine model of gut colonization were significantly lower after TEM-1 ß-lactamase overproduction and lower still after E240G substitution and OmpK36 loss. We hypothesize that the chosen courses of antibiotics led to the stepwise emergence of a clone with resistance to penicillins and ESCs and no loss of virulence. However, acquired resistance may have a fitness cost that limits evolutionary success. Our results might explain why the overproduction of extended-spectrum ß-lactamases (which should confer a high level of piperacillin-tazobactam resistance) is not observed in clinical practice and why TEM-207 has rarely been detected in clinical isolates.

Clinical emergence of a novel extended-spectrum variant deriving from the OXY-1 ß-lactamase.

The genomic comparison of two Klebsiella michiganensis clinical isolates recovered from the same patient, one resistant to piperacillin-tazobactam and intermediate to cefotaxime, the other resistant to ceftazidime but susceptible to piperacillin-tazobactam, revealed one mutation in the blaOXY-1-24 gene accounting for a L169M substitution in the Ω loop. Cloning experiment in Escherichia coli demonstrated the contribution of this mutation to the hydrolysis spectrum extension towards ceftazidime and cefepime, whereas the resistance to piperacillin-tazobactam was reduced. To the best of our knowledge, this study shows for the first time that ceftazidime resistance can occur in vivo from OXY-1 precursor by structural alteration.

Frameshift mutation (690delG) in cpxA contributes to the extensive drug resistance of a Serratia marcescens clinical isolate.

OBJECTIVES: To identify the genetic change responsible for resistance to penicillins, extended-spectrum cephalosporins (ESCs), aminoglycosides and ciprofloxacin in a Serratia marcescens clinical isolate recovered from a pancreatic abscess 6 weeks after a WT strain was isolated from the same patient. The impact on the fitness was also assessed. METHODS: The genomes of both S. marcescens isolates were sequenced using Illumina technology, assembled, annotated and compared with each other. PCR amplification followed by Sanger sequencing was carried out to confirm the mutation. Complementation of the resistant isolate with a recombinant plasmid harbouring the WT gene was performed. The growth rates were measured for both isolates in LB medium. RESULTS: Comparative genomic analysis disclosed only one frameshift mutation (690delG) in the cpxA gene, which codes for the histidine kinase of a two-component system (TCS). This change introduced a premature termination codon, leading to the truncated CpxA_HatR variant that contained 234 amino acids instead of 464. Complementation, which consisted of transfer of the WT cpxA into the resistant S. marcescens derivative, restored completely its susceptibility to ESCs, aminoglycosides and ciprofloxacin, thus confirming the contribution of the CpxA_HatR variant to resistance. Growth analysis showed that the fitness of the resistant isolate was unchanged. CONCLUSIONS: This study shows for the first time that constitutive activation of the Cpx pathway can per se confer resistance to ESCs and ciprofloxacin, in addition to the aminoglycoside resistance usually described. It sheds new light on the role of altered TCSs in fostering bacterial survival.

Orogenital Transmission of Neisseria meningitidis Causing Acute Urethritis in Men Who Have Sex with Men.

Neisseria meningitidis sequence type 11 is an emerging cause of urethritis. We demonstrate by using whole-genome sequencing orogenital transmission of a N. meningitidis sequence type 11 isolate causing urethritis in a monogamous couple of men who have sex with men. These results suggest dissemination of this clonal complex among low-risk patients.

Structural Alteration of OmpR as a Source of Ertapenem Resistance in a CTX-M-15-Producing Escherichia coli O25b:H4 Sequence Type 131 Clinical Isolate.

In this study, an ertapenem-nonsusceptible Escherichia coli isolate was investigated to determine the genetic basis for its carbapenem resistance phenotype. This clinical strain was recovered from a patient that received, 1 year previously, ertapenem to treat a cholangitis due to a carbapenem-susceptible extended-spectrum-ß-lactamase (ESBL)-producing E. coli isolate. Whole-genome sequencing of these strains was performed using Illumina and single-molecule real-time sequencing technologies. It revealed that they belonged to the ST131 clonal group, had the predicted O25b:H4 serotype, and produced the CTX-M-15 and TEM-1 ß-lactamases. One nucleotide substitution was identified between these strains. It affected the ompR gene, which codes for a regulatory protein involved in the control of OmpC/OmpF porin expression, creating a Gly-63-Val substitution. The role of OmpR alteration was confirmed by a complementation experiment that fully restored the susceptibility to ertapenem of the clinical isolate. A modeling study showed that the Gly-63-Val change displaced the histidine-kinase phosphorylation site. SDS-PAGE analysis revealed that the ertapenem-nonsusceptible E. coli strain had a decreased expression of OmpC/OmpF porins. No significant defect in the growth rate or in the resistance to Dictyostelium discoideum amoeba phagocytosis was found in the ertapenem-nonsusceptible E. coli isolate compared to its susceptible parental strain. Our report demonstrates for the first time that ertapenem resistance may emerge clinically from ESBL-producing E. coli due to mutations that modulate the OmpR activity.

Extended-spectrum ß-lactamases and plasmid-mediated quinolone resistance in enterobacterial clinical isolates from neonates in Tunisia.

This study was conducted to investigate extended-spectrum-ß-lactamase (ESBL) producing Enterobacteriaceae isolates from the Center of Maternity and Neonatology of Monastir, Tunisia. Fourty-six strains out of 283 were found to produce ESBL: Klebsiella pneumoniae (n = 37), Escherichia coli (n = 6), Enterobacter cloacae (n = 2), and Citrobacter freundi (n = 1). Genotyping analysis, using ERIC2 and RAPD, showed that strains were clonally unrelated. PCR amplification followed by sequencing revealed that all strains produced CTX-M-15. This enzyme was co-produced with TEM and SHV determinants in 34 and 36 strains respectively. The blaCTXM-15 gene was bracked by ISEcp1 and/or IS26 in 42 out of the 46 ESBL positive strains. The quinolone resistance determinants were associated to the ESBL producing isolates: we identified the qnrB1 gene in six isolates and the aac(6')-Ib-cr gene in five isolates. This epidemiological study shows the widespread of CTX-M-15 and qnr determinants among enterobacterial isolates from neonates hospitalized at the center of Maternity and Neonatology of Monastir suggesting either mother portage or horizontal transmission.

Molecular Characterization of Carbapenem-Nonsusceptible Enterobacterial Isolates Collected during a Prospective Interregional Survey in France and Susceptibility to the Novel Ceftazidime-Avibactam and Aztreonam-Avibactam Combinations.

An interregional surveillance program was conducted in the northwestern part of France to determine the prevalence of carbapenem-nonsusceptible Enterobacteriaceae (CNSE) isolates and their susceptibility to ceftazidime-avibactam and aztreonam-avibactam combinations. Nonduplicate CNSE clinical isolates were prospectively collected from six hospitals between June 2012 and November 2013. MICs of ceftazidime and aztreonam, alone or combined with a fixed concentration of avibactam (4 µg/ml), and those of carbapenems (comparator agents) were determined. MICs of ertapenem in combination with phenylalanine arginine-naphthylamide dihydrochloride (PAßN) were also determined to assess active efflux. Genes encoding carbapenemases, plasmid-mediated AmpC enzymes, extended-spectrum ß-lactamases (ESBLs), and major outer membrane proteins (OMPs) were amplified and sequenced. OMPs were also extracted for SDS-PAGE analysis. Among the 139 CNSE isolates, mainly Enterobacter spp. and Klebsiella pneumoniae, 123 (88.4%) were ertapenem nonsusceptible, 12 (8.6%) exhibited reduced susceptibility to all carbapenems, and 4 Proteeae isolates (2.9%) were resistant to imipenem. Carbapenemase production was detected in only two isolates (producing OXA-48 and IMI-3). In contrast, OMP deficiency, in association with AmpCs and/or ESBLs (mainly CTX-M-9, SHV-12, and CTX-M-15), was largely identified among CNSE isolates. The ceftazidime-avibactam and aztreonam-avibactam combinations exhibited potent activity against CNSE isolates (MIC50/MIC90, 1/1 µg/ml and 0.5/0.5 µg/ml, respectively) compared to that of ceftazidime and aztreonam alone (MIC50/MIC90, 512/512 µg/ml and 128/512 µg/ml, respectively). This study reveals the in vitro activity of ceftazidime-avibactam and aztreonam-avibactam combinations against a large collection of porin-deficient enterobacterial isolates that are representative of the CNSE recovered in the northern part of France.

Characterization of a novel AmpC ß-lactamase produced by a carbapenem-resistant Cedecea davisae clinical isolate.

A Cedecea davisae isolate, which was intermediate or resistant to third-generation cephalosporins and carbapenems, was recovered from a urine sample. Susceptibility testing, isoelectric focusing, and analysis of outer membrane proteins showed that AmpC ß-lactamase expression combined with porin deficiency accounted for the carbapenem resistance. A cloning experiment followed by phenotypic and enzymatic characterization identified a novel class C enzyme that was phylogenetically and biochemically close to the chromosome-borne ß-lactamases of the genera Enterobacter and Citrobacter.

Comparison of Xpert MRSA/SA Nasal and MRSA/SA ELITe MGB assays for detection of the mecA gene with susceptibility testing methods for determination of methicillin resistance in Staphylococcus aureus isolates.

In a series of 82 Staphylococcus strains isolated from culture, 100% were identified as Staphylococcus aureus by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS); 99.9% (77/82) of them were resistant to benzylpenicillin, oxacillin, and cefoxitin, and 6.1% (5/82) were susceptible to methicillin. Xpert MRSA/SA assay results were concordant with the phenotypic results in 76.8% (63/82) of cases and discordant in 23.2% (19/82) of cases. The MRSA/SA ELITe MGB kit results were concordant with phenotypic results in 100% of the cases. When comparing the Xpert MRSA/SA assay results with the MRSA/SA ELITe MGB kit results, 78% (64/82) of the cases were concordant, while 22% (18/82) of the cases were discordant. No statistically significant differences were observed between the two techniques. The PCR protocol that was used to validate the results of these two methods gave the following results: 49 were conventional methicillin-resistant S. aureus (MRSA) isolates (mecA positive and mecALGA251 negative), and 25 were phenotypic MRSA isolates (mecA negative and mecALGA251 positive).

Hydrolysis spectrum extension of CMY-2-like ß-lactamases resulting from structural alteration in the Y-X-N loop.

The Citrobacter freundii isolate CHA, which was responsible for postoperative peritonitis after 10 days of cefepime therapy, displayed a phenotype of resistance consistent with extended-spectrum AmpC (ESAC) ß-lactamase. The chromosome-borne bla(AmpC-CHA) gene was amplified and sequenced, revealing five amino acid substitutions, I125V, R148H, Q196H, V305A, and V348A, in the product compared to the sequence of native AmpC. A cloning experiment yielded the Escherichia coli TOP10(pAmpC-CHA) strain, which was resistant to all extended-spectrum cephalosporins (ESCs), including cefepime. To ascertain whether the R148H substitution accounted for the hydrolysis spectrum extension, it was reverted by site-directed mutagenesis. The resulting E. coli TOP10(pAmpC-CHA-H148R) strain was fully susceptible to cefepime, thus confirming that the Arg-148 replacement was mandatory for substrate profile enlargement. To further characterize the phenotypical and biochemical effects induced by the R148H change, it was introduced by site-directed mutagenesis into the CMY-2 ß-lactamase, which is structurally related to the chromosome-borne cephalosporinase of C. freundii. The CMY-2-R148H variant conferred increased MICs of ESCs, whereas those of carbapenems were unchanged even in a porin-deficient E. coli strain. Moreover, it exhibited increased catalytic efficiency (k(cat)/K(m)) toward ceftazidime (100-fold) due to an enhanced hydrolysis rate (k(cat)), whereas the enzymatic parameters toward imipenem were unchanged. The structural analysis of the AmpC variant showed that the R148H replacement occurred in the loop containing the Y-X-N motif, which is the counterpart of the SDN loop in class A ß-lactamases. This study shows that the Y-X-N loop is a novel hot spot for mutations accounting for hydrolysis spectrum extension in CMY-2-type enzymes.

Emergence of ertapenem resistance in an Escherichia coli clinical isolate producing extended-spectrum beta-lactamase AmpC.

Escherichia coli isolate MEV, responsible for a bloodstream infection, was resistant to penicillins, cephalosporins, and ertapenem. Molecular and biochemical characterization revealed the production of a novel, chromosome-borne, extended-spectrum AmpC (ESAC) ß-lactamase with a Ser-282 duplication and increased carbapenemase activity. This study demonstrates for the first time that chromosome-borne ESAC ß-lactamases can contribute to the emergence of ertapenem resistance in E. coli clinical isolates.

Sensitive and specific phenotypic assay for metallo-beta-lactamase detection in Enterobacteria by use of a moxalactam disk supplemented with EDTA.

Moxalactam is highly hydrolyzed by plasmid-mediated metallo-ß-lactamases (MBLs), whereas it is poorly inactivated by serine-active carbapenemases. This study demonstrated that moxalactam resistance constituted an effective screen for MBL expression in enterobacteria, which could be confirmed, even in low-MBL-producing isolates, by a disk potentiation test using moxalactam and EDTA.

Emergence of Imipenem Resistance in a CpxA-H208P-Variant-Producing Proteus mirabilis Clinical Isolate.

The Proteus mirabilis PmirS clinical isolate, which was susceptible to imipenem (0.5 µg/mL) and amikacin (1 µg/mL), was recovered from a bronchial aspirate of a patient who recently underwent lung transplantation. The P. mirabilis PmirR clinical isolate, which exhibited resistance to imipenem (16 µg/mL) and amikacin (24 µg/mL), was isolated 3 weeks later from the same patient and the same specimen type. Using short-read sequencing technology, these isolates appeared to be genetically identical except the cpxA gene of the PmirR isolate that was mutated leading to the His-208-Pro substitution. The structural alteration was localized in the histidine kinase, adenylate cyclase, methyl accepting protein, phosphatase (HAMP) domain, which is involved in the signal transduction between the sensor kinase and the regulator response of the CpxA/CpxR two-component system (TCS). No significant defect in the growth rate was found between the PmirS and PmirR isolates. This study suggests that alteration in CpxA might confer imipenem and amikacin resistance in P. mirabilis. This study brings new evidence that the TCS alteration could provide an adaptive capacity in a clinical context by conferring antibiotic resistance without fitness cost.

Phenotypic and biochemical comparison of the carbapenem-hydrolyzing activities of five plasmid-borne AmpC ß-lactamases.

The CMY-2, ACT-1, DHA-1, ACC-1, and FOX-1 enzymes are representative of five plasmid-mediated AmpC (pAmpC) ß-lactamase clusters. Resistance to imipenem has been reported in Enterobacteriaceae as a result of pAmpC expression combined with decreased outer membrane permeability. The aim of this study was to determine the role of different pAmpCs in carbapenem resistance and to define the structure/activity relationship supporting carbapenemase activity. The ampC genes encoding the five pAmpCs and the chromosomal AmpC of Escherichia coli EC6, which was used as a reference cephalosporinase, were cloned and introduced into wild-type E. coli TOP10 and OmpC/OmpF porin-deficient E. coli HB4 strains. The MICs of ß-lactams for the recombinant strains revealed that CMY-2, ACT-1, and DHA-1 ß-lactamases conferred a high level of resistance to ceftazidime and cefotaxime once expressed in E. coli TOP10 and reduced significantly the susceptibility to imipenem once expressed in E. coli HB4. In contrast, FOX-1 and ACC-1 enzymes did not confer resistance to imipenem. Biochemical analysis showed that CMY-2 ß-lactamase and, to a lesser extent, ACT-1 exhibited the highest catalytic efficiency toward imipenem and showed low K(m) values. A modeling study revealed that the large R2 binding site of these two enzymes may support the carbapenemase activity. Therefore, CMY-2-type, ACT-1-type, and DHA-1-type ß-lactamases may promote the emergence of carbapenem resistance in porin-deficient clinical isolates.

Characterization of CSP-1, a novel extended-spectrum beta-lactamase produced by a clinical isolate of Capnocytophaga sputigena.

The Capnocytophaga sputigena isolate NOR, responsible for septicemia, was resistant to amoxicillin and narrow-spectrum cephalosporins. In a cloning experiment, a new gene, bla(CSP-1), was identified; this gene encodes a novel extended-spectrum beta-lactamase (ESBL) that shares only 52% and 49% identities with the CME-1 and VEB-1 beta-lactamases, respectively. The G+C content of this gene, its genetic environment, the absence of conjugation transfer, and its detection in two reference strains suggested that it was an intrinsic resistance gene located on the chromosome.

Contribution to Carbapenem Resistance and Fitness Cost of DcuS/DcuR, RcsC/RcsB, and YehU/YehT Two-Component Systems in CTX-M-15-Producing Escherichia coli.

Alteration in two-component systems (TCSs), which are signal transduction pathways in prokaryotes, can result in antibiotic resistance. Recently, it has been shown that the overexpression, using a multicopy cloning vector, of the dcuR, rcsB, and yehT genes, which code for the response regulator (RR) part of TCSs, enhanced the minimal inhibitory concentrations (MICs) of carbapenems in Escherichia coli K-12 derivative KAM3. Herein, the contribution to carbapenem resistance of the DcuS/DcuR, RcsC/RcsB, and YehU/YehT TCSs was assessed in E. coli K-12 derivative BW25113 (A phylogroup) and 536 (B2 phylogroup) recipient strains in combination with extended-spectrum ß-lactamase that exhibit a weak carbapenemase activity. The genes encoding both the sensor kinase (SK) and the RR, on the one hand, and the genes encoding the SK only, on the other hand, of these regulating pathways were disrupted. Subsequently, the mutants and their parental strains were transformed by a recombinant plasmid encoding the CTX-M-15 gene, before testing their susceptibility to carbapenems and their fitness. Results showed a trade-off between enhanced MICs for ertapenem, which remained above the clinical resistance breakpoint, and decreased growth rate, specifically for the 536 strain SK mutants. In conclusion, mutations in dcuS/dcuR, rcsC/rcsB, and yehU/yehT genes may be a pivotal first-step event in the development of carbapenem resistance.

Role of the Ser-287-Asn replacement in the hydrolysis spectrum extension of AmpC beta-lactamases in Escherichia coli.

Two AmpC variants harboring the S287N substitution were obtained by mutagenesis from cephalosporinases representative of the phylogenetic groups A and B2 of Escherichia coli. Their biochemical characterization revealed that the S287N replacement led to an important increase in the catalytic efficiency toward extended-spectrum cephalosporins in the AmpC beta-lactamase of group A only.