AmpC Hyperproduction Research Articles

Clonal Distribution and Its Association With the Carbapenem Resistance Mechanisms of Carbapenem-Non-Susceptible Pseudomonas aeruginosa Isolates From Korean Hospitals.

Carbapenem resistance in Pseudomonas aeruginosa is a serious global health problem. We investigated the clonal distribution and its association with the carbapenem resistance mechanisms of carbapenem-non-susceptible P. aeruginosa isolates from three Korean hospitals. A total of 155 carbapenem-non-susceptible P. aeruginosa isolates collected between 2011 and 2019 were analyzed for sequence types (STs), antimicrobial susceptibility, and carbapenem resistance mechanisms, including carbapenemase production, the presence of resistance genes, OprD mutations, and the hyperproduction of AmpC β-lactamase. Sixty STs were identified in carbapenem-non-susceptible P. aeruginosa isolates. Two high-risk clones, ST235 (N=41) and ST111 (N=20), were predominant; however, sporadic STs were more prevalent than high-risk clones. The resistance rate to amikacin was the lowest (49.7%), whereas that to piperacillin was the highest (92.3%). Of the 155 carbapenem-non-susceptible isolates, 43 (27.7%) produced carbapenemases. Three metallo-β-lactamase (MBL) genes, blaIMP-6 (N=38), blaVIM-2 (N=3), and blaNDM-1 (N=2), were detected. blaIMP-6 was detected in clonal complex 235 isolates. Two ST773 isolates carried blaNDM-1 and rmtB. Frameshift mutations in oprD were identified in all isolates tested, regardless of the presence of MBL genes. Hyperproduction of AmpC was detected in MBL gene-negative isolates. Frameshift mutations in oprD combined with MBL production or hyperproduction of AmpC are responsible for carbapenem resistance in P. aeruginosa. Further attention is required to curb the emergence and spread of new carbapenem-resistant P. aeruginosa clones.

Serratia marcescens enzyme SME-2 isolated from sputum in New Zealand.

The Serratia marcescens enzymes (SMEs) are chromosomally encoded Ambler Class A carbapenem-hydrolysing β-lactamases, which distinctively express resistance to carbapenems while remaining susceptible to extended-spectrum cephalosporins. Global reports of SMEs are infrequent. Here we describe the isolation of an SME-2-producing S. marcescens from the sputum of a patient who was hospitalized at Christchurch Hospital, New Zealand. An immunosuppressed asthmatic patient who presented with shortness of breath and hypoxia grew S. marcescens from a sputum culture. Antimicrobial susceptibilities were determined by Phoenix, with MICs of meropenem and imipenem determined by Liofilchem® MIC gradient strips and interpreted according to EUCAST breakpoints. Investigation for carbapenemase was performed using Carba NP, modified CIM (mCIM) and GeneXpert® Carba-R. WGS was performed using the Illumina DNA Prep library kit and sequenced using MiSeq. The isolate showed an unusual susceptibility profile, including high-level resistance to meropenem and imipenem, while remaining susceptible to extended-spectrum cephalosporins. The Carba NP and mCIM were positive and WGS demonstrated the presence of a blaSME-2 gene located on the chromosome within the SmarGI1-1 genomic island. In addition, a blaSRT-like class C β-lactamase, aac(6')-Ic aminoglycoside-modifying enzyme and various multidrug efflux mechanisms were found. Phylogenetic core-genome analysis indicated no matching genome with RefSeq database strains. S. marcescens is an opportunistic pathogen of concern, harbouring a variety of intrinsic resistance mechanisms, including the potential for stable AmpC hyperproduction. Globally, SME-type carbapenemases have been infrequently reported; however, isolates carrying this mechanism could have limited treated options, having implications for patient management. To the best of our knowledge this is the first report of SME in New Zealand.

Bloodstream Infections by AmpC-Producing Enterobacterales: Risk Factors and Therapeutic Outcome.

Bloodstream infections associated with AmpC-producing Enterobacterales are severe medical conditions which, without prompt and effective treatment, may have dire ramifications. This study aimed to assess whether certain comorbidities and previous surgical procedures coincide with resistance determinants of AmpC-producing Enterobacterales associated with bloodstream infections. Antibiotic resistance patterns and therapy outcome were also determined. The patients' data obtained revealed that the prevalence of recent surgical procedures, solid organ tumors, metabolic diseases, kidney and liver failure, and hematological malignancies do not differ between resistant and susceptible isolates of AmpC-producing Enterobacterales. Furthermore, no difference was reported in mortality rates. Regarding antibiotic resistance, 34.52% of isolates were confirmed to be resistant (AmpC hyperproduction, ESBL, or carbapenemase). More than one in five AmpC hyperproducers were reported amid Providencia spp., K. aerogenes, E. cloacae, and C. freundii. strains. Carbapenemases were mostly noted in Providencia spp. followed by M. morganii and K. aerogenes strains. Serratia marcescens had the highest proportion of ESBLsof ESBLs. Resistance to expanded-spectrum cephalosporins of Providencia spp. and K. aerogenes strains exceeded 50%, and resistance to meropenem over 10% was observed only in C. freundii strains. Enterobacterales' ever-growing resistance to antibiotics is becoming quite a challenge for clinicians and new treatment options are required.

Hospital-acquired infections due to carbapenem-resistant Providencia stuartii.

During the course of a retrospective survey on healthcare associated infections (HAIs) due to carbapenem-resistant organisms, an unusual prevalence of HAIs due to carbapenem-resistant Providencia stuartii (CRPS) was found. Hence this study aimed to conduct the occurrence of P. stuartii associated HAIs with special reference to the drug resistance profiling of these isolates. Of the eight total HAI cases (7.5% of total HAIs and 33.3% of HAIs due to Enterobacterales) of CRPS infections included in this study, three were reported from ventilator-associated pneumonia (VAP), three were surgical site infections (SSIs), one was a catheter-associated urinary tract infection (CAUTI) and one was a bloodstream infection. All the eight CRPS isolates were tested for extended-spectrum β-lactamases production, AmpC hyperproduction as well as carbapenem resistance. Typing of the isolates was performed by repetitive extragenic palindromic polymerase chain reaction (REP-PCR). All the eight isolates of CRPS were found to be AmpC hyperproducers, carbapenemase producers, and harboured chromosomally located blaNDM in seven isolates and blaIMP genes in one. All the cases with CRPS infections had prior history of colistin therapy along with prolonged hospital stay (>20 days). The cases were located in five different wards/intensive care unit (ICU) within the hospital in one year. However, strain typing by REP-PCR revealed 100 per cent similarity and clonal relatedness in all the seven isolates carrying blaNDM genes. Interestingly, routine hospital surveillance revealed a high carriage of P. stuartii in the axilla of patients admitted to the ICU. The study findings suggest CRPS as an important cause of HAIs. This organism often goes unnoticed due to the burden of carbapenem resistance in other Enterobacterales and non-fermenters.

426. Trends in carbapenem non-susceptible (Carb-NS) Enterobacterales from hospitalized patients in the US from 2014-2020: A comparison between organisms with and without concerns for chromosomal ampC beta-lactamase hyperproduction

Abstract Background Carbapenem-resistant Enterobacterales (ENT) were designated an “Urgent Threat” by the CDC, underscoring need for understanding epidemiological trends over time with these pathogens. We evaluated trends in non-susceptibility to carbapenems (Carb-NS) in ENT in the inpatient setting from 2014-2020 across 314 US facilities nation-wide, with a focus on comparisons between ENT with and without concerns for chromosomal ampC beta-lactamase hyperproduction. Table:Model-estimated inpatient-setting Carb NS % and trends from 2014-2020.Figure:Trends in inpatient % Carb-NS ENT from 2014-2020. Methods All adults with a positive ENT culture (first isolate of a species per 30-day period from blood, respiratory, urine, skin/wound, intra-abdominal, or other) in the inpatient setting from 314 US hospitals from 2014 - 2020 were evaluated (BD Insights Research Database, Becton, Dickinson & Company). Carb-NS was defined as intermediate or resistant to ertapenem, imipenem, meropenem or doripenem per commercial panels. Organisms were classified as concerning for hyperproduction of chromosomal ampC b-lactamase (K. aerogenes, E. cloacae, C. freundii; ampC-ENT) and those without such concerns (E coli [EC], K. pneumoniae [KPN], P mirabilis [PM]); non-ampC ENT). Other ENT were excluded. Time series models were used to evaluate the monthly patterns of resistance trends in proportion per isolates tested. Results 2,513,392 inpatient non-duplicate ENT isolates were evaluated across 314 facilities of which 31,628 (1.3%) were Carb-NS. For Carb-NS ENT, 60.9% (19,277/31,628) were from non-ampC ENT and 30.7% (9,717/31,628) were in ampC ENT; together, these groups made up 91.6% of ENT evaluated. There were significant decreases in overall trends in inpatient ENT by % Carb-NS and % Carb-NS KPN (p < 0.001 for both). There were significant increases (see Table) in inpatient % Carb NS for PM and ampC-ENT (p < 0.0001 for both). Conclusion There was significant decrease in overall inpatient % Carb-NS ENT and % Carb-NS KPN from 2014-2020. However, significant increases in % Carb-NS were seen in organisms concerning for hyperproduction of chromosomal ampC. PM, in particular, should be further evaluated for genotypic resistance. Surveillance of Carb-NS isolates should include a broader set of pathogens beyond KPN and EC. Disclosures Vikas Gupta, PharmD, Becton, Dickinson and Company: Employee of, and shareholder in, Becton, Dickinson and Company, and the company received funding from GlaxoSmithKline plc. to conduct this study|GlaxoSmithKline plc.: GlaxoSmithKline plc.-sponsored study 212502 Kalvin Yu, MD, FIDSA, Becton, Dickinson and Company: Employee of, and shareholder in, Becton, Dickinson and Company, and the company received funding from GlaxoSmithKline plc. to conduct this study|GlaxoSmithKline plc.: GlaxoSmithKline plc.-sponsored study 212502 jason M. Pogue, PharmD, consultant to Venatorx, Merck, Shionogi, QPex Biopharma, Utility, GSK, and Entasis.: Advisor/Consultant Janet Watts, PhD, Becton, Dickinson and Company: Employee of Becton, Dickinson and Company, and the company received funding from GlaxoSmithKline plc. to conduct this study|GlaxoSmithKline plc.: GlaxoSmithKline plc.-sponsored study 212502 Cornelius J. Clancy, MD, receives research funding paid to his institution from Astellas and Merck: Grant/Research Support|serves as an advisory Board member for Astellas, Cidara, and Scynexis, served on the advisory board for Merck, Qpex Biopharma, and Shionogi: Advisor/Consultant|Venatorx and Needham & Associates: Advisor/Consultant.

Evaluation of an Antibiotic Susceptibility Testing Method on Enterobacterales-Positive Blood Cultures in Less Than 8 h Using the Rapid Mueller-Hinton Diffusion Method in Conjunction with the SIRscan 2000 Automatic Reading Device.

Enterobacterales bloodstream infections are life-threatening and require rapid, targeted antibiotherapy based on antibiotic susceptibility testing (AST). A new method using Muller-Hinton Rapid-SIR (MHR-SIR) agar (i2a, Montpellier, France) allows complete direct AST (dAST) to be read from positive blood culture bottles (BCBs) for all Enterobacterales species after 6–8 h of incubation. We evaluated (i) the performance of dAST from positive BCBs on MHR-SIR agar using two different inoculum protocols; (ii) the categorical agreement between dAST results obtained with MHR-SIR agar vs. those obtained with Muller-Hinton (MH) agar; and (iii) the ability of the MHR-SIR medium to detect β-lactam resistant Enterobacterales. Finally, we estimated the saved turnaround time (TAT) with MHR-SIR compared with MH agar in our 24/7 laboratory. Our results showed that the most suitable inoculation protocol for dAST on MHR-SIR agar was 1 drop of BCB/5 mL H2O. For monomicrobial Enterobacterales BCBs, dAST performed on MHR-SIR medium showed 99.3% categorical agreement with AST on MH agar. Furthermore, MHR-SIR agar allows early detection of β-lactam resistance mechanisms, including AmpC hyperproduction, extended-spectrum β-lactamase, and carbapenemase. Finally, TAT reduction in our 24/7 laboratory was 16 h, enabling a significantly faster provision of antibiotic advice.

Detection and Characterization of Carbapenemases in Enterobacterales With a New Rapid and Simplified Carbapenemase Detection Method Called rsCDM.

ObjectiveThis study aimed to develop a new rapid and simplified carbapenemase detection method (rsCDM) for detection and characterization of carbapenemase with 3-aminophenylboronic acid (APBA), ethylenediaminetetraacetic acid (EDTA), and cloxacillin (CLO) β-lactamase inhibitors.MethodsA panel of 182 carbapenem-resistant Enterobacterales (CRE) strains with blaKPC (88), blaNDM (60), blaIMP (10), blaVIM (3), blaOXA-181 (5), blaKPC, and blaNDM (7), porin changes in combination with an extended-spectrum β-lactamase (ESBL) (3) or AmpC hyper-production (6) and 43 carbapenem-susceptible Enterobacterales isolates were used to evaluate the performance of rsCDM and EDTA-carbapenem inactivation method (eCIM). Carbapenemase class was determined with specific inhibitors at 4, 6, and 18 h by rsCDM, and the difference between imipenem (IMI) and meropenem (MEM) disks was simultaneously compared.ResultsThe sensitivity of rsCDM using IMI was 97.1% at the three time points, with a specificity of 100%, independent of the culture duration. Similar to IPM, MEM disk also showed high sensitivity (97.1%) and specificity (100%) at 6 h. And the sensitivity of eCIM was 95.4% and the specificity was 100%. Based on a decision algorithm, the characterization number of IMI and MEM in KPC-producing isolates was 88 vs. 87, metallo-β-lactamases (MBLs) was 73 vs. 72, KPC and NDM carbapenemase was 7 vs. 7 at 4 h, respectively. After 6 h, the category number changed insignificantly except for isolates with combined AmpC overproduction and porin changes, showing an increase in IMI (6) and MEM (2), and there was no difference in the results between 6 and 18 h for the two tablets. OXA-181-producing strains can’t be distinguished by rsCDM. For eCIM, the characterization number in KPC-, OXA- 181-, and MBLs-producing strains was 88, 5, and 72, but it failed to detect multi-enzyme-producing isolates (KPC and NDM).ConclusionrsCDM accurately discriminated carbapenemase within 4 h and could differentiate multi-enzyme (KPC and NDM) and AmpC in conjunction with porin changes strains. Hence, rsCDM represents a rapid, simple, easy readout, and accurate tool that can be used without any specialized equipment.

AmpC hyperproduction in a Cedecea davisae implant-associated bone infection during treatment: a case report and therapeutic implications

BackgroundData on antimicrobial resistance mechanisms are scanty for Cedecea spp., with very variable antibiotic resistance patterns documented. Here we report the first in vivo resistance evolution of a C. davisae clinical isolate in a patient with a complex hand trauma and provide insight in the resistance mechanism, leading to therapeutic implications for this pathogen.Case presentationCedecea davisae was isolated from a patient with hand trauma during a first surgical debridement. Six days after primary surgical treatment and under antimicrobial treatment with amoxicillin-clavulanic acid and later cefepime, follow up cultures yielded C. davisae which demonstrated a resistance development. The susceptible parental isolate and its resistant derivative were characterized by whole genome sequencing, ampC, ompC and ompF by RT- PCR. The resistant derivative demonstrated an A224G SNP in ampD, the transcriptional regulator of ampC, leading to a His75Arg change in the corresponding AmpD protein. AmpC transcription of the resistant derivative was 362-times higher than the susceptible isolate. Transcription levels of ompF and ompC were 8.5-fold and 1.3-fold lower, respectively, in the resistant derivative. Downregulation of OmpF putatively resulted from a mutation in the presumed promoter region upstream of the dusB-Fis operon, a proposed regulator for ompF.ConclusionsThis case demonstrates the in vivo resistance development of C. davisae within 7 days similar to that of the members of the Enterobacter cloacae complex. Our findings add valuable information for future therapeutic management of these opportunistic pathogens as they warrant the same empirical treatment as AmpC producers.

Genome-wide analysis in Escherichia coli unravels a high level of genetic homoplasy associated with cefotaxime resistance.

Cefotaxime (CTX) is a third-generation cephalosporin (3GC) commonly used to treat infections caused by Escherichia coli . Two genetic mechanisms have been associated with 3GC resistance in E. coli . The first is the conjugative transfer of a plasmid harbouring antibiotic-resistance genes. The second is the introduction of mutations in the promoter region of the ampC β-lactamase gene that cause chromosome-encoded β-lactamase hyperproduction. A wide variety of promoter mutations related to AmpC hyperproduction have been described. However, their link to CTX resistance has not been reported. We recultured 172 cefoxitin-resistant E. coli isolates with known CTX minimum inhibitory concentrations and performed genome-wide analysis of homoplastic mutations associated with CTX resistance by comparing Illumina whole-genome sequencing data of all isolates to a PacBio sequenced reference chromosome. We mapped the mutations on the reference chromosome and determined their occurrence in the phylogeny, revealing extreme homoplasy at the −42 position of the ampC promoter. The 24 occurrences of a T at the −42 position rather than the wild-type C, resulted from 18 independent C>T mutations in five phylogroups. The −42 C>T mutation was only observed in E. coli lacking a plasmid-encoded ampC gene. The association of the −42 C>T mutation with CTX resistance was confirmed to be significant (false discovery rate <0.05). To conclude, genome-wide analysis of homoplasy in combination with CTX resistance identifies the −42 C>T mutation of the ampC promotor as significantly associated with CTX resistance and underlines the role of recurrent mutations in the spread of antibiotic resistance.

Heteroresistance to cefepime in Pseudomonas aeruginosa bacteraemia

IntroductionHeteroresistance to antibiotic agents can lead to diagnostic and therapeutic failures; however, to date, cefepime heteroresistance (FEP-HR) in Pseudomonas aeruginosa (P. aeruginosa) bacteraemia has not been characterised. The primary goal of this study was to investigate the molecular epidemiology, mechanisms and risk factors for cefepime-heteroresistant P. aeruginosa bacteraemia over approximately 6 years in Southwest China. ResultsA high prevalence (57.3%) of heteroresistance to cefepime was observed during the study period, and these FEP-HR isolates were not clonally related. Mechanistic studies revealed that AmpC hyperproduction contributed to the development of this phenomenon. In addition, patients with advanced age, haematological malignancies, central venous catheters, and previous cephalosporin therapy were identified as independent risk factors for acquiring FEP-HR P. aeruginosa bacteraemia. Furthermore, patients infected with FEP-HR were generally at a greater risk for an adverse prognosis compared with those with non-FEP-HR. More importantly, characterisation of three successive P. aeruginosa isolates recovered from the same patient revealed that heteroresistance can act as an intermediate stage during the evolution from susceptibility to full resistance in patients undergoing antibiotic therapy for prolonged periods. ConclusionThese findings emphasised the necessity of antimicrobial stewardship programs in clinical settings, as well as the need for some rapid screening methods for detecting this phenomenon.

Regulation of AmpC-Driven β-Lactam Resistance in Pseudomonas aeruginosa: Different Pathways, Different Signaling.

The hyperproduction of the chromosomal AmpC β-lactamase is the main mechanism driving β-lactam resistance in Pseudomonas aeruginosa, one of the leading opportunistic pathogens causing nosocomial acute and chronic infections in patients with underlying respiratory diseases. In the current scenario of the shortage of effective antipseudomonal drugs, understanding the molecular mechanisms mediating AmpC hyperproduction in order to develop new therapeutics against this fearsome pathogen is of great importance. It has been accepted for decades that certain cell wall-derived soluble fragments (muropeptides) modulate AmpC production by complexing with the transcriptional regulator AmpR and acquiring different conformations that activate/repress ampC expression. However, these peptidoglycan-derived signals have never been characterized in the highly prevalent P. aeruginosa stable AmpC hyperproducer mutants. Here, we demonstrate that the previously described fragments enabling the transient ampC hyperexpression during cefoxitin induction (1,6-anhydro-N-acetylmuramyl-pentapeptides) also underlie the dacB (penicillin binding protein 4 [PBP4]) mutation-driven stable hyperproduction but differ from the 1,6-anhydro-N-acetylmuramyl-tripeptides notably overaccumulated in the ampD knockout mutant. In addition, a simultaneous greater accumulation of both activators appears linked to higher levels of AmpC hyperproduction, although our results suggest a much stronger AmpC-activating potency for the 1,6-anhydro-N-acetylmuramyl-pentapeptide. Collectively, our results propose a model of AmpC control where the activator fragments, with qualitative and quantitative particularities depending on the pathways and levels of β-lactamase production, dominate over the repressor (UDP-N-acetylmuramyl-pentapeptide). This study represents a major step in understanding the foundations of AmpC-dependent β-lactam resistance in P. aeruginosa, potentially useful to open new therapeutic conceptions intended to interfere with the abovementioned cell wall-derived signaling.IMPORTANCE The extensive use of β-lactam antibiotics and the bacterial adaptive capacity have led to the apparently unstoppable increase of antimicrobial resistance, one of the current major global health challenges. In the leading nosocomial pathogen Pseudomonas aeruginosa, the mutation-driven AmpC β-lactamase hyperproduction stands out as the main resistance mechanism, but the molecular cues enabling this system have remained elusive until now. Here, we provide for the first time direct and quantitative information about the soluble cell wall-derived fragments accounting for the different levels and pathways of AmpC hyperproduction. Based on these results, we propose a hierarchical model of signals which ultimately govern ampC hyperexpression and resistance.

Characterization of cefotaxime-resistant urinary Escherichia coli from primary care in South-West England 2017-18.

Third-generation cephalosporin-resistant Escherichia coli from community-acquired urinary tract infections are increasingly reported worldwide. We sought to determine and characterize the mechanisms of cefotaxime resistance employed by urinary E. coli obtained from primary care, over 12 months, in Bristol and surrounding counties in South-West England. Cefalexin-resistant E. coli isolates were identified from GP-referred urine samples using disc susceptibility testing. Cefotaxime resistance was determined by subsequent plating onto MIC breakpoint plates. β-Lactamase genes were detected by PCR. WGS was performed on 225 isolates and analyses were performed using the Center for Genomic Epidemiology platform. Patient information provided by the referring general practices was reviewed. Cefalexin-resistant E. coli (n=900) isolates were obtained from urines from 146 general practices. Following deduplication by patient approximately 69% (576/836) of isolates were cefotaxime resistant. WGS of 225 isolates identified that the most common cefotaxime-resistance mechanism was blaCTX-M carriage (185/225), followed by plasmid-mediated AmpCs (pAmpCs) (17/225), AmpC hyperproduction (13/225), ESBL blaSHV variants (6/225) or a combination of both blaCTX-M and pAmpC (4/225). Forty-four STs were identified, with ST131 representing 101/225 isolates, within which clade C2 was dominant (54/101). Ciprofloxacin resistance was observed in 128/225 (56.9%) of sequenced isolates, predominantly associated with fluoroquinolone-resistant clones ST131 and ST1193. Most cefalexin-resistant E. coli isolates were cefotaxime resistant, predominantly caused by blaCTX-M carriage. The correlation between cefotaxime resistance and ciprofloxacin resistance was largely attributable to the high-risk pandemic clones ST131 and ST1193. Localized epidemiological data provide greater resolution than regional data and can be valuable for informing treatment choices in the primary care setting.

Development of an algorithm to discriminate between plasmid- and chromosomal-mediated AmpC β-lactamase production in Escherichia coli by elaborate phenotypic and genotypic characterization.

ObjectivesAmpC-β-lactamase production is an under-recognized antibiotic resistance mechanism that renders Gram-negative bacteria resistant to common β-lactam antibiotics, similar to the well-known ESBLs. For infection control purposes, it is important to be able to discriminate between plasmid-mediated AmpC (pAmpC) production and chromosomal-mediated AmpC (cAmpC) hyperproduction in Gram-negative bacteria as pAmpC requires isolation precautions to minimize the risk of horizontal gene transmission. Detecting pAmpC in Escherichia coli is challenging, as both pAmpC production and cAmpC hyperproduction may lead to third-generation cephalosporin resistance.MethodsWe tested a collection of E. coli strains suspected to produce AmpC. Elaborate susceptibility testing for third-generation cephalosporins, WGS and machine learning were used to develop an algorithm to determine ampC genotypes in E. coli. WGS was applied to detect pampC genes, cAmpC hyperproducers and STs.ResultsIn total, 172 E. coli strains (n=75 ST) were divided into a training set and two validation sets. Ninety strains were pampC positive, the predominant gene being blaCMY-2 (86.7%), followed by blaDHA-1 (7.8%), and 59 strains were cAmpC hyperproducers. The algorithm used a cefotaxime MIC value above 6 mg/L to identify pampC-positive E. coli and an MIC value of 0.5 mg/L to discriminate between cAmpC-hyperproducing and non-cAmpC-hyperproducing E. coli strains. Accuracy was 0.88 (95% CI=0.79–0.94) on the training set, 0.79 (95% CI=0.64–0.89) on validation set 1 and 0.85 (95% CI=0.71–0.94) on validation set 2.ConclusionsThis approach resulted in a pragmatic algorithm for differentiating ampC genotypes in E. coli based on phenotypic susceptibility testing.

English

Escherichia coli present the ampC naturally, and the observation of phenotypical resistance to cefoxitin is related to this gene deregulation. Mutations in the regulatory region in ampC cause exaggerated expression. The most frequent alterations in the E. coli AmpC promoter/attenuator leading to this overexpression is described at the positions: -88, -82, -42, -18, -1 and +58. Mastitis studies were carried in Rio de Janeiro and Mato Grosso, Brazil. Two cefoxitin and amoxicillin-clavulanic acid-resistant E. coli from farms animals were unusually detected once these characteristics are not observed together in this species. The objective of this work was to determine if these isolates had a chromosomal gene mutation, determining AmpC hyperproduction. After DNA sequencing, mutations were observed at -88, -82, -73, -18, -1 and +58 positions, confirming the initially suspected AmpC hyperexpression. In Brazil, this is the first work to report E. coli hyperproducing this enzyme. &nbsp; Key words: Ampc attenuator, AmpC hyperproduction, ampC promoter, bovine feces, mastitic milk.

AmpD homologs in biotypes of Yersinia enterocolitica: Implications in regulation of chromosomal AmpC-type cephalosporinases

Inducible ‘AmpC-type’ chromosomal cephalosporinases have been reported to be differentially expressed in different biotypes of Yersinia entercolocolitica. AmpD amidases are key regulators of the expression of ampC genes in Y. entercolocolitica as their inactivation results in hyper production of AmpC. To understand the differences in regulation of ampC expression in different biotypes of Y. enterocolitica, characteristics of ampD homologs were studied in strains of Y. enterocolitica belonging to five biotypes namely 1A, 1B, 2, 3 and 4. Our results indicated that the mechanisms which regulate expression of ampC might differ in different biotypes. While a three-step regulation mechanism seemed to be functional in biotypes 2, 3 and 4, a two-step regulation mechanism using other AmiD like proteins might be functional in biotypes 1A and 1B. The existence of ampD homolog(s)-mediated expression of ampC in other members of the family Enterobacteriaceae may provide further credence to our findings.

Emergence of blaVEB and blaGES among VIM-producing Pseudomonas aeruginosa clinical isolates in Alexandria, Egypt.

Thirty-three Pseudomonas aeruginosa isolates, resistant to one or more β-lactams, were included in this study. Identification of tested strains was confirmed using MALDI-TOF/MS. Phenotypic and genotypic β-lactamase patterns were investigated. Most of the isolates were resistant to carbapenems (32 out of 33) and to the extended-spectrum cephalosporins (ESC) (30 out of 33). Phenotypically, the production of extended-spectrum beta-lactamase (ESBL), metallo-β-lactamases (MBL), and carbapenemases was detected in 10, 23, and 9 isolates, respectively. However, AmpC hyperproduction was not phenotypically detected among all isolates. Genotypically, ESBL and MBL encoding genes were detected in 23 and 27 isolates, respectively. Altogether 27 strains were detected as blaVIM positive and 16 strains carried blaOXA-10 gene. To the best of our knowledge, this is the first report of P. aeruginosa clinical isolates harboring blaVEB together with blaGES in Egypt, where 5 of our 30 ESC-resistant isolates showed this genotype. Our results confirmed that resistance of P. aeruginosa isolates to β-lactam antibiotics is mediated via multiple β-lactamases belonging to different molecular classes. To the best of our knowledge, this is the first report of blaVEB among P. aeruginosa clinical isolates from Egypt. Ten isolates harbored blaVEB and five of them co-harbored blaVEB together with blaGES, blaVIM, and blaOXA-10.

Role of Low-Molecular-Mass Penicillin-Binding Proteins, NagZ and AmpR in AmpC β-lactamase Regulation of Yersinia enterocolitica.

Yersinia enterocolitica encodes a chromosomal AmpC β-lactamase under the regulation of the classical ampR-ampC system. To obtain a further understanding to the role of low-molecular-mass penicillin-binding proteins (LMM PBPs) including PBP4, PBP5, PBP6, and PBP7, as well as NagZ and AmpR in ampC regulation of Y. enterocolitica, series of single/multiple mutant strains were systematically constructed and the ampC expression levels were determined by luxCDABE reporter system, reverse transcription-PCR (RT-PCR) and β-lactamase activity test. Sequential deletion of PBP5 and other LMM PBPs result in a continuously growing of ampC expression level, the β-lactamse activity of quadruple deletion strain YEΔ4Δ5Δ6Δ7 (pbp4, pbp5, pbp6, and pbp7 inactivated) is approached to the YEΔD123 (ampD1, ampD2, and ampD3 inactivated). Deletion of nagZ gene caused two completely different results in YEΔD123 and YEΔ4Δ5Δ6Δ7, NagZ is indispensable for YEΔ4Δ5Δ6Δ7 ampC derepression phenotype but dispensable for YEΔD123. AmpR is essential for ampC hyperproduction in these two types of strains, inactivation of AmpR notable reduced the ampC expression level in both YEΔD123 and YEΔ4Δ5Δ6Δ7.

Novel sequence types of extended-spectrum and acquired AmpC beta-lactamase producing Escherichia coli and Escherichia clade V isolated from wild mammals.

The closer contact with wildlife due to the growing human population and the destruction of natural habitats emphasizes the need of gaining insight into the role of animals as source of antimicrobial resistance. Here, we aim at characterizing the antimicrobial resistance genes and phylogenetic distribution of commensal Escherichia coli from 62 wild mammals. Isolates exhibiting resistance to ≥1 antibiotic were detected in 25.8% of the animals and 6.4% carried an extended-spectrum beta-lactamase (ESBL)/AmpC-producing E. coli. Genetic mechanisms involved in third-generation cephalosporin resistance were as follows: (i) hyperproduction of chromosomal AmpC (hedgehog), (ii) production of acquired CMY-2 β-lactamase (hedgehog), (iii) production of SHV-12 and CTX-M-14 ESBLs (n =2, mink and roe-deer). ESBL genes were transferable by conjugation, and blaCMY-2 was mobilized by a 95kb IncI1 plasmid. The distribution of the phylogenetic groups in the E. coli collection studied was B1 (44.6%), B2 (24.6%), E (15.4%), A (4.6%) and F (3.1%). Five isolates (7.7%) were cryptic Escherichia clades (clade IV, 4 mice; clade V, 1 mink). ESBL/AmpC-E. coli isolates showed different sequence types (STs): ST1128/B1, ST4564/B1 (new), ST4996/B1 (new) and a non-registered ST. This study contributes to better understand the E. coli population and antimicrobial resistance flow in wildlife and reports new AmpC-E. coli STs and a first described ESBL-producing Escherichia clade V isolate.

Molecular epidemiological survey of bacteremia by multidrug resistant Pseudomonas aeruginosa: the relevance of intrinsic resistance mechanisms.

The bacterial factors associated with bacteremia by multidrug-resistant and extensively drug-resistant P. aeruginosa, including overexpression of efflux pumps, AmpC overproduction, and loss/alteration of the OprD porin in isolates that are non-Metallo-β-Lactamase producing were analyzed in a retrospective study. Molecular analyses included strain typing by Pulsed Field Gel Electrophoresis and identification of key genes via qualitative and quantitative PCR-based assays. Previous use of carbapenems and tracheostomy was independently associated with the development of bacteremia by extensively drug-resistant and multidrug-resistant strains of P. aeruginosa. A high consumption of antimicrobials was observed, and 75.0% of the isolates contained amplicons with the blaSPM-1 and blaVIM genes. Of the 47 non-Metallo-β-Lactamase isolates, none had another type of carbapenemase. However, the isolates exhibited high rates of hyperproduction of AmpC, loss of the OprD porin (71.4%) and the presence of MexABOprM (57.1%) and MexXY (64.3%). This study suggests that in non-Metallo-β-Lactamase isolates, the association of intrinsic resistance mechanisms could contributes to the expression of multidrug-resistant/extensively drug-resistant phenotypes.

Ceftolozane-Tazobactam Resistance in Multidrug-Resistant Pseudomonas aeruginosa Isolates Not Associated with AmpC Activity

Background Ceftolozane-tazobactam (CT) is a newly approved cephalosporin/β-lactamase inhibitor combination with excellent in vitro activity against multidrug-resistant (MDR) P. aeruginosa. Unfortunately, CT-resistance (CT-R) has already been reported. In this work, we evaluate mutational pathways associated with high level of CT-R and assess the role of AmpC in a clinical strain-pair of MDR P. aeruginosa.Methods A CT susceptible isolate of P. aeruginosa (2365) and its CT-R derivatives (2366 and 2367) were recovered from the infected device of a patient before and after treatment with CT. Minimum inhibitory concentrations (MICs) to CT were determined by Etest. Resistance mediated by AmpC hyperproduction was evaluated using ceftazidime (CAZ) and meropenem (MER) with and without cloxacillin (CLOX) at concentration of 1 mg/ml. In addition, the β-lactamase hydrolysis activity was determined for crude cell lysate of the isolates using a spectrophotometric assay for nitrocefin degradation. Furthermore, whole genome sequencing of the three strains was performed and compared (2365 vs. 2366 and 2367). Reads from each isolate were mapped against the genome of the reference strain PAO1. Variants identified by GATK, SamTools and CLC Genomics Workbench 8.5 were selected and annotated with SnpEff.ResultsStrain 2365 had a CT MIC of 0.75 mg/ml while 2366 and 2367 have MICs > 256 mg/ml. AmpC hyperproduction test was positive only for the susceptible isolate (2365). In concordance, the hydrolysis assay showed a lack of nitrocefin degradation by CT-R 2366 compared with its CT-susceptible isolate 2365. Notably, the three strains (S and R) exhibited a truncated AmpD. Comparison of the resistant derivatives vs. 2365 and 2367 showed a 7 amino acid deletion in the Ω-loop of the β-lactamase AmpC in both resistant derivatives and mutations in genes predicted to encode a hypothetical protein, an ABC transporter ATP-binding protein and a multidrug resistance operon repressor MexR.Conclusion Our results suggest that the deletion in the Ω-loop of AmpC in 2366 and 2367 does not contribute to CT-R in these P. aeruginosa strains. Further characterization of AmpC and other predicted proteins identified by WGS are needed to determine the mechanism of CT-R.Disclosures All authors: No reported disclosures.