Chromosomal Class Research Articles

Molecular Characterization of Serratia marcescens Strain Isolated from Yellow Mealworms, Tenebrio molitor, in The Netherlands.

Insect culture has developed rapidly worldwide; it faces important security and safety control issues, including animal infections and disease development. In the Netherlands, in 2021, a ~30% mortality of mealworms, Tenebrio molitor, occurred at one farm, where over-humid sites in the substrate were observed. Bacterial cultures from both the external and internal partsof fry and larger mealworms were identified by MALDI-TOF to predominantly Serratia marcescens, Staphylococcus xylosus and Staphylococus saprofyticus. Due to the important role of S. marcescens as a potential zoonotic bacterium, we performed a molecular characterization of the isolated strain. Genomic analysis showed a multidrug-resistant S. marcescens isolate carrying a tet (41), aac (6')-Ic, and blaSST-1 chromosomal class C beta-lactamase-resistantgenes, all located on the chromosome. Additionally, several virulence genes were identified. The phylogenetic tree revealed that the S. marcescens strain from this study was similar to other S. marcescens strains from different ecological niches. Although the entomopathogenic activity was not confirmed, this case demonstrates that T. molitor can act as a reservoir and as an alternative path for exposing clinically important antibiotic-resistant bacteria that can affect animals and humans. It underlines the need to keep management factors optimal, before insects and their products enter the feed and food chain.

Relebactam restores susceptibility of resistant Pseudomonas aeruginosa and Enterobacterales and enhances imipenem activity against chromosomal AmpC-producing species: analysis of global SMART 2018–2020

BackgroundCarbapenem-resistant bacteria are an increasing problem in clinical practice; thus, it is important to identify β-lactamase inhibitors (e.g., relebactam) that can restore carbapenem susceptibility. We report analyses of relebactam enhancement of imipenem activity against both imipenem-nonsusceptible (NS) and imipenem-susceptible (S) Pseudomonas aeruginosa and Enterobacterales. Gram-negative bacterial isolates were collected for the ongoing Study for Monitoring Antimicrobial Resistance Trends global surveillance program. Clinical and Laboratory Standards Institute–defined broth microdilution minimum inhibitory concentrations (MIC) were used to determine the imipenem and imipenem/relebactam antibacterial susceptibilities of P. aeruginosa and Enterobacterales isolates.ResultsBetween 2018 and 2020, 36.2% of P. aeruginosa (N = 23,073) and 8.2% of Enterobacterales (N = 91,769) isolates were imipenem-NS. Relebactam restored imipenem susceptibility in 64.1% and 49.4% of imipenem-NS P. aeruginosa and Enterobacterales isolates, respectively. Restoration of susceptibility was largely observed among K. pneumoniae carbapenemase-producing Enterobacterales and carbapenemase-negative P. aeruginosa. Relebactam also caused a lowering of imipenem MIC among imipenem-S P. aeruginosa and Enterobacterales isolates from chromosomal Ambler class C β-lactamase (AmpC)–producing species. For both imipenem-NS and imipenem-S P. aeruginosa isolates, relebactam reduced the imipenem MIC mode from 16 μg/mL to 1 μg/mL and from 2 μg/mL to 0.5 μg/mL, respectively, compared with imipenem alone.ConclusionsRelebactam restored imipenem susceptibility among nonsusceptible isolates of P. aeruginosa and Enterobacterales and enhanced imipenem susceptibility among susceptible isolates of P. aeruginosa and isolates from Enterobacterales species that can produce chromosomal AmpC. The reduced imipenem modal MIC values with relebactam may result in a higher probability of target attainment in patients.

Evaluation of Phenotypic Tests to Detect Extended-Spectrum β-Lactamase (ESBL)-Producing Klebsiella oxytoca Complex Strains.

Klebsiella oxytoca complex (KoC) species may overproduce their chromosomal class A OXY β-lactamases, conferring reduced susceptibility to piperacillin-tazobactam, expanded-spectrum cephalosporins and aztreonam. Moreover, since clavulanate maintains its ability to inhibit these enzymes, the resulting resistance phenotype may falsely resemble the production of acquired extended-spectrum β-lactamases (ESBLs). In this work, a collection of 44 KoC strains of human and animal origin was characterized with whole-genome sequencing (WGS) and broth microdilution (BMD) susceptibility testing. Comparison of ESBL producers (n = 11; including CTX-M-15 [n = 6] and CTX-M-1 [n = 5] producers) and hyperproducers of OXYs (n = 21) showed certain phenotypic differences: piperacillin-tazobactam (MIC90s: 16 versus >64 μg/mL), cefotaxime (MIC90s: 64 versus 4 μg/mL), ceftazidime (MIC90s: 32 versus 4 μg/mL), cefepime (MIC90s: 8 versus 4 μg/mL) and associated resistance to non-β-lactams (e.g., trimethoprim-sulfamethoxazole: 90.9% versus 14.3%, respectively). However, a clear phenotype-based distinction between the two groups was difficult. Therefore, we evaluated 10 different inhibitor-based confirmatory tests to allow such categorization. All tests showed a sensitivity of 100%. However, only combination disk tests (CDTs) with cefepime/cefepime-clavulanate and ceftazidime/ceftazidime-clavulanate or the double-disk synergy test (DDST) showed high specificity (100%, 95.5%, and 100%, respectively). All confirmatory tests in BMD or using the MIC gradient strip did not perform well (specificity, ≤87.5%). Of note, ceftazidime/ceftazidime-avibactam tests also exhibited low specificity (CDT, 87.5%; MIC gradient strip, 77.8%). Our results indicate that standard antimicrobial susceptibility profiles can raise some suspicion, but only the use of cefepime/cefepime-clavulanate CDT or DDST can guarantee distinction between ESBL-producing KoC strains and those hyperproducing OXY enzymes.

Characterization of Two Novel AmpC Beta-Lactamases from the Emerging Opportunistic Pathogen, Cedecea neteri

The genus Cedecea (family Enterobacteriaceae) causes a wide spectrum of acute infections in immunocompromised hosts, from pneumonia and bacteremia to oral ulcers and dialysis-related peritonitis. While Cedecea infections are reported infrequently in the literature, documented clinical cases of this emerging opportunistic human pathogen have occurred worldwide. Cedecea neteri has clinical significance and exhibits antimicrobial drug resistance. However, little is known about the molecular basis underlying the resistance phenotypes in C. neteri. We previously hypothesized that the open-reading frame cnt10470 in the C. neteri SSMD04 genome encodes a chromosomal Ambler class C (AmpC) β-lactamase based on sequence homology. In this study, recombinant polyhistidine-tagged proteins were created by cloning the putative ampC genes from SSMD04 and C. neteri ATCC 33855 (a clinical isolate) into the pET-6xHN expression vector, overexpressing the proteins, and then purifying the recombinant AmpCs (rAmpCs) using immobilized metal affinity chromatography (Ni-NTA). The in vitro enzymatic analysis of the purified rAmpCs was performed to determine the Km and kcat for various β-lactam substrates. The rAmpCs are functional class C β-lactamases when assayed using the chromogenic β-lactamase substrate, nitrocefin. The presence of functional AmpCs in both C. neteri strains underscores the necessity of performing antibiotic susceptibility testing in the management of C. neteri infections.

Characterization and identification of a novel chromosomal class C β-lactamase, LAQ-1, and comparative genomic analysis of a multidrug resistance plasmid in Lelliottia amnigena P13.

Lelliottia amnigena, a bacterium usually isolated from natural environments, may cause human infections and has been suggested to be naturally resistant to second- and third-generation cephalosporins. In this study, we determined the whole-genome sequence of an isolate, L. Amnigena P13, isolated from animal farm sewage. On the basis of genome sequence analysis, susceptibility testing, molecular cloning, and enzyme kinetic parameter analysis, we identified a novel chromosome-encoded AmpC β-lactamase, LAQ-1. bla LAQ-1 is resistant to penicillin G, ampicillin, and several first- to fourth-generation cephalosporins, such as cefazolin, cefoxitin and cefepime. The MIC levels of some β-lactams, such as cefoxitin, cefepime, aztreonam and cefazolin, for the recombinant clone (pUCP24-bla LAQ-1/DH5α) increased by approximately 4- to 64-fold compared with those of the control strain (pUCP24/DH5α). The kinetic properties of LAQ-1, with the highest catalytic activity observed toward piperacillin, were basically the same as those of typical class C β-lactamases, and avibactam had a strong inhibitory effect on its hydrolytic activity. The genetic background of bla LAQ-1 was relatively conserved, and no mobile genetic element (MGE) was found around it. The plasmid pP13-67 of L. amnigena P13 harbored 12 resistance genes [qnrS1, aph(6)-Id, aadA2, sul1, sul2, bla TEM-1, qacEΔ1, dfrA12, tetA and floR] related to different mobile genetic elements within an ~22 kb multidrug resistance region. The multidrug resistance region shared the highest nucleotide sequence similarities with those of the chromosomes or plasmids of different bacterial species, indicating the possibility of horizontal transfer of these resistance genes among different bacterial species.

Sex in Symbiodiniaceae dinoflagellates: genomic evidence for independent loss of the canonical synaptonemal complex

Dinoflagellates of the Symbiodiniaceae family encompass diverse symbionts that are critical to corals and other species living in coral reefs. It is well known that sexual reproduction enhances adaptive evolution in changing environments. Although genes related to meiotic functions were reported in Symbiodiniaceae, cytological evidence of meiosis and fertilisation are however yet to be observed in these taxa. Using transcriptome and genome data from 21 Symbiodiniaceae isolates, we studied genes that encode proteins associated with distinct stages of meiosis and syngamy. We report the absence of genes that encode main components of the synaptonemal complex (SC), a protein structure that mediates homologous chromosomal pairing and class I crossovers. This result suggests an independent loss of canonical SCs in the alveolates, that also includes the SC-lacking ciliates. We hypothesise that this loss was due in part to permanently condensed chromosomes and repeat-rich sequences in Symbiodiniaceae (and other dinoflagellates) which favoured the SC-independent class II crossover pathway. Our results reveal novel insights into evolution of the meiotic molecular machinery in the ecologically important Symbiodiniaceae and in other eukaryotes.

Isolation and characterisation of carbapenem-resistant Xanthomonas citri pv. mangiferaeindicae-like strain gir from the faecal material of giraffes.

The purpose of this study was to determine if giraffes (Giraffa camelopardalis) living in captivity at the Jacksonville Zoo and Gardens, Jacksonville, FL were colonised with carbapenem-resistant bacteria and, if found, to identify underlying genetic mechanisms contributing to a carbapenem-resistant phenotype. Faecal samples from seven giraffes were examined for carbapenem-resistant bacteria. Only one isolate (a Xanthomondaceae) was found to be carbapenem-resistant by antibiotic susceptibility testing. This isolate was selected for additional characterization, including whole genome sequencing (WGS). Based on average nucleotide identity, the bacterium was identified as Xanthomonas citri pv. mangiferaeindicae-like strain gir. Phenotypic carbapenemase tests and PCR for the most common carbapenemase genes produced negative results, suggesting that carbapenem resistance was mediated by another mechanism. Resistance gene profile analysis of WGS results confirmed these results. Among identified resistance genes, a chromosomal class A beta-lactamase with 71% identity to the penP beta-lactamase gene from Xanthomonas citri ssp. citri was identified, which could contribute to a carbapenem-resistant phenotype.

Activity of imipenem/relebactam against carbapenemase-producing Enterobacteriaceae with high colistin resistance.

Imipenem/relebactam, an investigational β-lactam/β-lactamase inhibitor combination for treatment of Gram-negative infections, and comparators including ceftazidime/avibactam, piperacillin/tazobactam and colistin were tested for activity against representative carbapenemase-producing Enterobacteriaceae (CPE) isolates. MICs of the antimicrobial agents were determined using standard broth microdilution methodology for CPE isolates collected from Indiana patients, primarily during the time frame of 2013-17 (n = 199 of a total of 200 isolates). Inhibitors were tested at 4 mg/L in all combinations. Of the CPE in the study, 199 produced plasmid-encoded KPC class A carbapenemases; 1 Serratia marcescens isolate produced the SME-1 chromosomal class A carbapenemase. MIC50/MIC90 values of imipenem/relebactam were ≤0.25/0.5 mg/L, whereas MIC50/MIC90 values of ceftazidime/avibactam were 1/2 mg/L. Resistance to colistin was observed in 54% (n = 97) of 180 non-Serratia isolates tested (MIC50 of 4 mg/L). Colistin resistance mechanisms included production of a plasmid-encoded mcr-1-like gene (n = 2) or an inactivated mgrB gene. Imipenem/relebactam was the most potent agent tested against CPE in this study and may be a useful addition to the antimicrobial armamentarium to treat infections caused by these pathogens.

Extensively Drug-Resistant Pseudomonas aeruginosa ST309 Harboring Tandem Guiana Extended Spectrum β-Lactamase Enzymes: A Newly Emerging Threat in the United States.

BackgroundTreatment of serious infections due to multidrug-resistant (MDR) Pseudomonas aeruginosa remains a challenge, despite the introduction of novel therapeutics. In this study, we report 2 extensively drug-resistant clinical isolates of sequence type (ST) 309 P aeruginosa resistant to all β-lactams, including the novel combinations ceftolozane/tazobactam, ceftazidime/avibactam, and meropenem/vaborbactam.MethodsIsolates were sequenced using both short-read (Illumina) and long-read technology to identify resistance determinants, polymorphisms (compared with P aeruginosa PAO1), and reconstruct a phylogenetic tree. A pair of β-lactamases, Guiana extended spectrum β-lactamase (GES)-19 and GES-26, were cloned and expressed in a laboratory strain of Escherichia coli to examine their relative impact on resistance. Using cell lysates from E coli expressing the GES genes individually and in tandem, we determined relative rates of hydrolysis for nitrocefin and ceftazidime.ResultsTwo ST309 P aeruginosa clinical isolates were found to harbor the extended spectrum β-lactamases GES-19 and GES-26 clustered in tandem on a chromosomal class 1 integron. The presence of both enzymes in E coli was associated with significantly elevated minimum inhibitory concentrations to aztreonam, cefepime, meropenem, ceftazidime/avibactam, and ceftolozane/tazobactam, compared with those expressed individually. The combination of ceftazidime/avibactam plus aztreonam was active in vitro and used to achieve cure in one patient. Phylogenetic analysis revealed ST309 P aeruginosa are closely related to MDR strains from Mexico also carrying tandem GES.ConclusionsThe presence of tandem GES-19 and GES-26 is associated with resistance to all β-lactams, including ceftolozane/tazobactam. Phylogenetic analysis suggests that ST309 P aeruginosa may be an emerging threat in the United States.

Characterization of the emerging multidrug-resistant Salmonella enterica serovar Indiana strains in China

ABSTRACT Emergence of multidrug-resistant (MDR) Salmonella enterica serovar Indiana (S. Indiana), a dominant Salmonella serovar in China, has raised global awareness because the MDR S. Indiana also was rapidly emerged in other countries recently. To improve our understanding of underlying MDR mechanism and evolution of this emerging zoonotic pathogen, here we examined the standard ATCC51959 strain together with 19 diverse and representative Chinese S. Indiana strains by performing comprehensive microbiological, molecular, and comparative genomics analyses. The findings from S1-PFGE, plasmid origin analysis and Southern blotting suggested the MDR phenotype in the majority of isolates was associated with large integron-carrying plasmids. Interestingly, further in-depth analyses of two recently isolated, plasmid-free MDR S. Indiana revealed a long chromosomal class I integron (7.8 kb) that is not linked to the Salmonella Genome Island 1 (SGI1), which is rare. This unique chromosomal integron shares extremely high similarity to that identified in a MDR E. coli plasmid pLM6771 with respect to both genomic organization and sequence identity. Taken together, both plasmid and chromosomal integron I exist in the examined MDR S. Indiana strains. This timely study represents a significant step toward the understanding of molecular basis of the emerging MDR S. Indiana.

Molecular Analysis of a blaIMP-1-Harboring Class 3 Integron in Multidrug-Resistant Pseudomonas fulva.

A multidrug-resistant (MDR) Pseudomonas fulva strain was isolated in 2006 from a urine sample. The isolate harbored the blaIMP-1 gene, which was located in a chromosomal Tn402-like class 3 integron as a gene cassette array of aacA31-fosE-blaIMP-1 Two mutations in gyrA and one mutation in parC were detected in quinolone-resistance-determining regions (QRDRs). We report a full-length, novel, blaIMP-1-carrying class 3 integron. This integron, together with mutations in QRDRs, could have influenced the MDR phenotype.

The Novel β-Lactamase Inhibitor, ETX-2514, in Combination with Sulbactam Effectively Inhibits Acinetobacter baumannii

BackgroundMultidrug resistant (MDR) Acinetobacter sp. were deemed a “serious” health threat by the Centers for Disease Control and Prevention with a daunting 63% of infections being nearly untreatable. Underlying this challenging pathogen are the presence of a chromosomal class C β-lactamase, Acinetobacter-derived cephalosporinase (ADC), as well as the abundant prevalence of class D OXA β-lactamases that hydrolyze carbapenems in conjunction with a lack of potent β-lactamase inhibitors. Based on the ability of ETX2514, a rationally designed novel diazabicyclooctane inhibitor (Figure 1A), to inhibit class D β-lactamases as well as PBPs, we hypothesized that highly resistant clinical isolates of A. baumannii will demonstrate susceptibility to the sulbactam-ETX2514 combination and that A. baumannii β-lactamases, ADC-7 and OXA-58 will be readily inactivated by ETX2514.MethodsSusceptibility testing according to Clinical and Laboratory Standards Institute was performed for sulbactam ±4 mg/L ETX2514 using 72 A. baumannii strains. More than half of the isolates are MDR, have ≥ eight resistant determinants, contain an ADC β-lactamase, and have OXA-23 and OXA-58-like β-lactamases in the carbapenem resistant isolates. ADC-7 and OXA-58 β-lactamases were purified and characterized with ETX2514 by steady-state inhibition kinetics and Q-TOF mass spectrometry.ResultsThe A. baumannii strains demonstrated an MIC90 of 32 mg/L for sulbactam and 2 mg/L for the sulbactam-ETX-2514 combination. The addition of ETX2514 lowered the MIC50 from 8 to 1 mg/L (Figure 1B). ETX-2514 effectively inhibited purified OXA-58 (k2/K = 2.5 ± 0.3 × 105 M−1 s−1 and Ki apP = 0.39 ± 0.01 µM) and ADC-7 (k2/K =1.0 ± 0.1 × 106 M−1 s−1 and Ki app = 0.11 ± 0.04 µM). The two β-lactamases displayed similar dissociation constants (Kd =1 ± 0.1 nM), but ADC-7 possessed a faster dissociation rate (koff =8 ± 1 × 10−4 s−1 for ADC-7 and 1.6 ± 0.3 × 10−4 s−1for OXA-58). Using mass spectrometry, ADC-7 and OXA-58 were found to stably bind the ETX-2514 compound for up to 24 hours (Figure 1C).ConclusionETX2514 is a new β-lactamase inhibitor that is strikingly effective at restoring susceptibility to highly drug-resistant A. baumannii isolates when combined with sulbactam via inhibition of the ADC-7 and OXA-58 β-lactamases.Disclosures F. Van Den Akker, Entasis: Grant Investigator, Research grant Wockhardt: Grant Investigator, Research grant; K. M. Papp-Wallace, Entasis: Grant Investigator, Research grant Allecra: Grant Investigator, Research grant Merck: Grant Investigator, Research grant Roche: Grant Investigator, Research grant Allergan: Grant Investigator, Research grant; R. A. Bonomo, Entasis: Grant Investigator, Research grant Allecra: Grant Investigator, Research grant Wockhardt: Grant Investigator, Research grant Merck: Grant Investigator, Research grant Roche: Grant Investigator, Research grant GSK: Grant Investigator, Research grant Allergan: Grant Investigator, Research grant Shionogi: Grant Investigator, Research grant

Comparison of different methods for identification of species of the genus Raoultella: report of 11 cases of Raoultella causing bacteraemia and literature review

The genus Raoultella was excised from Klebsiella in 2001, but difficulties in its identification may have led to an underestimation of its incidence and uncertainty on its pathogenic role. Recently, clinical reports involving Raoultella have increased, probably through the introduction of mass-spectrometry in clinical microbiology laboratories and the development of accurate molecular techniques. We performed a retrospective analysis using our blood culture collection (2011–14) to identify Raoultella isolates that could have been erroneously reported as Klebsiella. PCR and gene sequencing of highly specific chromosomal class A β-lactamase genes was established as the reference method, and compared with 16S rRNA and rpoβ sequencing, as well as matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS), MicroScan Walkaway system and API20E biochemical identification. MALDI-TOF and rpoβ correctly identified all Raoultella isolates, whereas 16S rRNA provided inconclusive results, and MicroScan and API20E failed to detect this genus. The analysis of the clinical characteristics of all Raoultella bacteraemia cases reported in the literature supports the role of Raoultella as an opportunistic pathogen that causes biliary tract infections in elderly patients who suffer from some kind of malignancy or have undergone an invasive procedure. Two salient conclusions are that Raoultella shows tropism for the biliary tract and so its identification could help clinicians to suspect underlying biliary tract disease when bacteraemia occurs. Concomitantly, as most phenotypic identification systems are not optimized for the identification of Raoultella, the use of MALDI-TOF or additional phenotypic tests is recommended for the reliable identification of this genus.

Wide Spread Prevalence of β-Lactam Resistance among Bacterial Species Obtained from Non-Clinical Samples

The potential threats of bacterial resistance, especially widespread multi-drug resistance, are of major concern. The scenario of a patient dying from an infection caused by a multi-drug resistant organism is now a reality. Infections caused by resistant strains of bacteria are related to higher morbidity and mortality. β-Lactam antibiotics are a broad class of antibiotics, consisting of penicillin, cephalosporin, Monobactam, and carbapenems. β-lactam antibiotics are used to treat various infections. β-lactamases are enzymes responsible to provide resistance to β-lactam antibiotics. Among Gram-negative bacteria, the emergence of resistance to expanded-spectrum cephalosporin has been a major concern. Initially a limited number of bacterial species that could mutate to hyper produce their chromosomal class C β-lactamase were found but now, resistance appeared in large number of bacterial species due to the production of TEM- or SHV-type ESBLs. In our study non clinical samples were selected to study the prevalence of drug resistance. After placing the various generations of β-lactam drugs we found that they were also resistant up to 4th generation of β-lactam drugs. This study confirms the growing incidences of β-lactam resistant strains in bacterial species of community origin.Int J Appl Sci Biotechnol, Vol 3(2): 248-255 DOI: http://dx.doi.org/10.3126/ijasbt.v3i2.12480

Genetic, biochemical characterization and mutagenesis of the chromosomal class A β-lactamase of Raoultella (formerly Klebsiella) terrigena

BackgroundChromosomal class A β-lactamases have been characterized in Raoultella ornithinolytica and Raoultella planticola. The purpose of this study was to characterize that of Raoultella terrigena. Materials and methodsThe blaTER-1 gene of R. terrigena strain ATCC33257T was cloned (pACter-1) and sequenced. It was then used to detect the bla gene of strains BM 85 01 095 and SB2796. The hypermutable Escherichia coli strain AB1157 mutS::Tn10 was transformed with pACter-1 and mutants growing on plates containing>2mg/L ceftazidime were studied. Notably, the impact of mutations only observed in the promoter region on β-lactam resistance was assessed by site-directed mutagenesis experiments. ResultsR. terrigena strains ATCC33257T and BM 85 01 095 had the same bla gene and deduced protein (TER-1) whereas there were 3 substitutions in those of strain SB2796 (TER-2). Class A β-lactamases TER showed 78%, 69.9% and 38.7% identity with PLA or ORN, TEM-1 and KOXY, respectively. Compared with TEM-1, TER-1 and TER-2 showed 2 particular substitutions, Leu75Pro and Glu240Asn demonstrated to be involved in the inherent β-lactam resistance profile of R. terrigena. TER-1 (pI of 7.6) had a high activity against penicillin G and a significantly low one against amoxicillin. Substitution G/T observed in the -35 region of the blaTER gene harbored by strains growing in the presence of≥2mg/L ceftazidime was shown to be responsible for this growth. ConclusionTER is a new class A β-lactamase belonging to functional group 2b.

Structure of ADC-68, a novel carbapenem-hydrolyzing class C extended-spectrum β-lactamase isolated from Acinetobacter baumannii.

Outbreaks of multidrug-resistant bacterial infections have become more frequent worldwide owing to the emergence of several different classes of β-lactamases. In this study, the molecular, biochemical and structural characteristics of an Acinetobacter-derived cephalosporinase (ADC)-type class C β-lactamase, ADC-68, isolated from the carbapenem-resistant A. baumannii D015 were investigated. The blaADC-68 gene which encodes ADC-68 was confirmed to exist on the chromosome via Southern blot analysis and draft genome sequencing. The catalytic kinetics of β-lactams and their MICs (minimum inhibitory concentrations) for A. baumannii D015 and purified ADC-68 (a carbapenemase obtained from this strain) were assessed: the strain was resistant to penicillins, narrow-spectrum and extended-spectrum cephalosporins, and carbapenems, which were hydrolyzed by ADC-68. The crystal structure of ADC-68 was determined at a resolution of 1.8 Å. The structure of ADC-68 was compared with that of ADC-1 (a non-carbapenemase); differences were found in the central part of the Ω-loop and the C-loop constituting the edge of the R1 and R2 subsites and are close to the catalytic serine residue Ser66. The ADC-68 C-loop was stabilized in the open conformation of the upper R2 subsite and could better accommodate carbapenems with larger R2 side chains. Furthermore, a wide-open conformation of the R2-loop allowed ADC-68 to bind to and hydrolyze extended-spectrum cephalosporins. Therefore, ADC-68 had enhanced catalytic efficiency against these clinically important β-lactams (extended-spectrum cephalosporins and carbapenems). ADC-68 is the first reported enzyme among the chromosomal class C β-lactamases to possess class C extended-spectrum β-lactamase and carbapenemase activities.

Acquired Class D β-Lactamases.

The Class D β-lactamases have emerged as a prominent resistance mechanism against β-lactam antibiotics that previously had efficacy against infections caused by pathogenic bacteria, especially by Acinetobacter baumannii and the Enterobacteriaceae. The phenotypic and structural characteristics of these enzymes correlate to activities that are classified either as a narrow spectrum, an extended spectrum, or a carbapenemase spectrum. We focus on Class D β-lactamases that are carried on plasmids and, thus, present particular clinical concern. Following a historical perspective, the susceptibility and kinetics patterns of the important plasmid-encoded Class D β-lactamases and the mechanisms for mobilization of the chromosomal Class D β-lactamases are discussed.

Extended-spectrum -lactamase- and AmpC -lactamase-producing D-tartrate-positive Salmonella enterica serovar Paratyphi B from broilers and human patients in Belgium, 2008-10

To characterize the genetic determinants responsible for extended-spectrum cephalosporin (ESC) resistance of d-tartrate-positive Salmonella enterica subsp. enterica serovar Paratyphi B (serovar Paratyphi B dT+) strains that have emerged in poultry and humans in Belgium during 2008-10. The ESC resistance genes among non-redundant serovar Paratyphi B dT+ strains were determined using PCR and sequencing. ESC phenotypes were horizontally transferred by conjugation. Extended-spectrum β-lactamase (ESBL)- or AmpC-carrying plasmids were typed by PCR-based replicon typing, plasmid multilocus sequence typing and restriction fragment length polymorphism. The genetic relationship of ESC-resistant strains was assessed by XbaI PFGE and multilocus sequence typing. Since 2008, the proportion of serovar Paratyphi B dT+ strains from broiler origin has increased significantly to reach 36.5% in 2010. Among 95 non-duplicate serovar Paratyphi B dT+ strains, 35% were resistant to ESCs. At the same time, a few ESC-resistant serovar Paratyphi B dT+ strains from humans were also detected in Belgium. The most prevalent ESBL gene, blaCTX-M-1, and the AmpC cephalosporinase gene blaCMY-2 were identified on various conjugative IncI1 plasmids of different sequence types and with different additional non-β-lactam phenotypes. Interestingly, the blaCTX-M-2 gene was located on large multireplicon IncHI2/P plasmids. In addition, highly ESC-resistant strains contained both the ESBL CTX-M-2 and the AmpC CMY-2 encoded by the IncHI2/P and IncI1 plasmids, respectively. All ESC-resistant serovar Paratyphi B dT+ strains belonged to sequence type 28 and showed the common PFGE pattern X8, as well as the chromosomal class 2 integron cassette array dfrA1-sat2-aadA1 previously described in the European poultry-associated serovar Paratyphi B dT+ clonal population. This study showed that the clonal population of multidrug-resistant serovar Paratyphi B dT+, persisting in broilers in Belgium for the last decade, recently acquired various plasmid-borne ESC resistance determinants, constituting a major concern for public health. Further surveillance programmes and research are an absolute necessity to understand their epidemiology and to propose interventions to limit the spread of ESC- and multidrug-resistant Salmonella spp.

A kinetic analysis of the inhibition of FOX-4 -lactamase, a plasmid-mediated AmpC cephalosporinase, by monocyclic -lactams and carbapenems

Class C β-lactamases are prevalent among Enterobacteriaceae; however, these enzymes are resistant to inactivation by commercially available β-lactamase inhibitors. In order to find novel scaffolds to inhibit class C β-lactamases, the comparative efficacy of monocyclic β-lactam antibiotics (aztreonam and the siderophore monosulfactam BAL30072), the bridged monobactam β-lactamase inhibitor BAL29880, and carbapenems (imipenem, meropenem, doripenem and ertapenem) were tested in kinetic assays against FOX-4, a plasmid-mediated class C β-lactamase (pmAmpC). The FOX-4 β-lactamase was purified. Steady-state kinetics, electrospray ionization mass spectrometry (ESI-MS) and ultraviolet difference (UVD) spectroscopy were conducted using the β-lactam scaffolds described. The K(i) values for the monocyclic β-lactams against FOX-4 β-lactamase were 0.04 ± 0.01 μM (aztreonam) and 0.66 ± 0.03 μM (BAL30072), and the Ki value for the bridged monobactam BAL29880 was 8.9 ± 0.5 μM. For carbapenems, the Ki values ranged from 0.27 ± 0.05 μM (ertapenem) to 2.3 ± 0.3 μM (imipenem). ESI-MS demonstrated the formation of stable covalent adducts when the monocyclic β-lactams and carbapenems were reacted with FOX-4 β-lactamase. UVD spectroscopy suggested the appearance of different chromophoric intermediates. Monocyclic β-lactam and carbapenem antibiotics are effective mechanism-based inhibitors of FOX-4 β-lactamase, a clinically important pmAmpC, and provide stimulus for the development of new inhibitors to inactivate plasmidic and chromosomal class C β-lactamases.

The Rise of Carbapenem-Resistant Acinetobacter baumannii

Acinetobacter spp. are Gram-negative bacteria that have become one of the most difficult pathogens to treat. The species A. baumannii, largely unknown 30 years ago, has risen to prominence particularly because of its ability to cause infections in immunocompromised patients. It is now a predominant pathogen in many hospitals as it has acquired resistance genes to virtually all antibiotics capable of treating Gram-negative bacteria, including the fluoroquinolones and the cephalosporins. Some members of the species have accumulated these resistance genes in large resistance islands, located in a "hot-spot" within the bacterial chromosome. The only conventional remaining treatment options were the carbapenems. However, A. baumannii possesses an inherent class D β-lactamase gene (blaOXA-51-like) that can have the ability to confer carbapenem resistance. Additionally, mechanisms of carbapenem resistance have emerged that derive from the importation of the distantly related class D β-lactamase genes blaOXA-23 and blaOXA-58. Although not inducible, the expression of these genes is controlled by mobile promoters carried on ISAba elements. It has also been found that other resistance genes including the chromosomal class C β-lactamase genes conferring cephalosporin resistance are controlled in the same manner. Colistin is now considered to be the final drug capable of treating infections caused by carbapenem-resistant A. baumannii; however, strains are now being isolated that are resistant to this antibiotic as well. The increasing inability to treat infections caused by A. baumannii ensures that this pathogen more than ranks with MRSA or Clostridium difficile as a threat to modern medicine.