Direct Colorimetry of Imipenem Decomposition as a Novel Cost-Effective Method for Detecting Carbapenemase-Producing Enterobacteria.

In the absence of a molecule that would collectively inhibit both metallo-ß-lactamases and serine-reactive carbapenemases, containment of their genes is the main weapon currently available for confronting carbapenem resistance in hospitals. Cost-effective methodologies rapidly detecting carbapenemase-producing enterobacteria (CPE) would facilitate such measures. Herein, a low-cost CPE detection method was developed that was based on the direct colorimetry of the yellow shift caused by the accumulation of diketopiperazines-products of the acid-catalyzed imipenem oligomerization-induced by carbapenemase action on dense solutions of imipenem/cilastatin. The reactions were studied by spectrophotometry in the visible spectrum using preparations of ß-lactamases from the four molecular classes. The effects of various buffers on reaction mixtures containing the potent carbapenemases NDM-1 and NMC-A were monitored at 405 nm. Optimal conditions were used for the analysis of cell suspensions, and the assay was evaluated using 66 selected enterobacteria, including 50 CPE as well as 16 carbapenemase-negative strains overexpressing other ß-lactamases. The development of the yellow color was specific for carbapenemase-containing enzyme preparations, and the maximum intensity was achieved in acidic or unbuffered conditions in the presence of zinc. When applied on bacterial cell suspensions, the assay could detect CPE with 98% sensitivity and 100% specificity, with results being comparable to those obtained with the Carba NP technique. Direct colorimetry of carbapenemase-induced imipenem decomposition required minimum reagents while exhibiting high accuracy in detecting CPE. Therefore, it should be considered for screening purposes after further clinical evaluation. IMPORTANCE Currently, the spread of multidrug-resistant (MDR) carbapenemase-producing enterobacteria (CPE), mostly in the clinical setting, is among the most pressing public health problems worldwide. In order to effectively control CPE, use of reliable and affordable methods detecting carbapenemase genes or the respective ß-lactamases is of vital importance. Herein, we developed a novel method, based on a previously undescribed phenomenon, that can detect CPE with few reagents by direct colorimetry of bacterial suspensions and imipenem/cilastatin mixtures.

Detection of carbapenemase producing enterobacteria using an ion sensitive field effect transistor sensor.

The timely and accurate detection of carbapenemase-producing Enterobacterales (CPE) is imperative to manage this worldwide problem in an effective fashion. Herein we addressed the question of whether the protons produced during imipenem hydrolysis could be detected using an ion sensitive field effect transistor (ISFET). Application of the methodology on enzyme preparations showed that the sensor is able to detect carbapenemases of the NDM, IMP, KPC and NMC-A types at low nanomolar concentrations while VIM and OXA-48 responded at levels above 100 nM. Similar results were obtained when CPE cell suspensions were tested; NDM, IMP, NMC-A and KPC producers caused fast reductions of the output potential. Reduction rates with VIM-type and especially OXA-48 producing strains were significantly lower. Based on results with selected CPEs and carbapenemase-negative enterobacteria, a threshold of 10 mV drop at 30 min was set. Applying this threshold, the method exhibited 100% sensitivity for NDM, IMP and KPC and 77.3% for VIM producers. The OXA-48-positive strains failed to pass the detection threshold. A wide variety of carbapenemase-negative control strains were all classified as negative (100% specificity). In conclusion, an ISFET-based approach may have the potential to be routinely used for non OXA-48-like CPE detection in the clinical laboratory.

Evaluation of the performance of Acuitas® Resistome Test and the Acuitas Lighthouse® software for the detection of ß-lactamase-producing microorganisms.

OBJECTIVES: Given the international spread of multidrug-resistant Gram-negative bacteria the need for prompt and precise characterization of underlying resistance traits has evolved into the cornerstone of infection control strategies. Novel commercial molecular tests enable rapid simultaneous testing for multiple resistance genes. We aimed to evaluate the performance of OpGen's Acuitas® Resistome Test and the Acuitas Lighthouse® software. METHODS: The test is tailored towards detecting 46 ß-lactamase genes (SHV and TEM variants associated with wild-type penicillin resistance, extended-spectrum ß-lactamases [ESBLs], acquired AmpCs and carbapenemases) via a microfluidic polymerase chain reaction (PCR) array. In total 118 isolates part of the collection of the Bacteriology Laboratory of the Hellenic Pasteur Institute, specifically 96 enterobacterial isolates and 21 Acinetobacter baumannii, of divergent origins, with previously characterized ß-lactamase content, were tested. RESULTS: In the enterobacterial group all 69 carbapenemase genes of the KPC, VIM, NDM and OXA-48 types were correctly identified (sensitivity, specificity, positive predictive value [PPV] and negative predictive value [NPV] of 100%). Non-ESBL SHV enzymes, ESBLs (CTX-M, GES, VEB types) and acquired AmpC enzymes were also correctly characterized. Of the 35 SHV-ESBLs harboured, correct identification was possible in 32/35 isolates, with overall sensitivity, specificity, PPV and NPV for the Klebsiella pneumoniae group of 89.29%, 100%, 100% and 91.18%, respectively. For the A. baumannii group the test exhibited an overall sensitivity for carbapenemase detection of 96.55% and 100% PPV. CONCLUSIONS: The OpGen Acuitas Resistome Test is an efficient molecular tool that can identify resistance threats in health care institutions with high diagnostic accuracy and be integrated into targeted surveillance protocols.

Changes in the rates and population structure of methicillin-resistant Staphylococcus aureus (MRSA) from bloodstream infections: A single-centre experience (2000-2015).

OBJECTIVES: The aim of this study was to assess the rate of methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infections (BSIs) and the population structure of MRSA isolates recovered between 2000-2015 in a tertiary-care hospital in Athens, Greece. METHODS: Non-duplicate MRSA blood isolates recovered during the study period were examined. Antimicrobial susceptibility testing was performed by Kirby-Bauer and gradient strip methods. Carriage of PVL and mecA genes was examined by PCR. Genetic relatedness of the isolates was studied by SCCmec, spa and multilocus sequence typing. RESULTS: A total of 398 MRSA BSI cases were identified. A decreasing trend in incidence from 1.69/10 000 patient-days in 2000 to 1.39/10 000 patient-days in 2015 (P=0.038) and in prevalence from 64.7% to 36.4% (P=0.008), respectively, was observed, whereas the incidence of methicillin-susceptible S. aureus BSI increased. MRSA isolates exhibiting resistance to common antistaphylococcal agents (excluding glycopeptides and the newer antistaphylococcals) decreased from 84.8% in 2000 to 0% in 2011 and were progressively 'replaced' by more susceptible phenotypes. A strong association between antimicrobial resistance phenotype and molecular type was observed. The pandemic HA-MRSA clone ST239-III progressively declined in parallel with increasing isolation frequency of two clonal complexes (CCs): HA-MRSA CC5, with the majority of isolates belonging to ST5-II; and CA-MRSA CC80, represented mainly by ST80-IV-t044, PVL+. CONCLUSION: The decline in MRSA BSI rates observed in our institution was associated with changes in population structure of the organism. This decline may be related to biological properties of the prevailing MRSA clones.

attI1-Located Small Open Reading Frames ORF-17 and ORF-11 in a Class 1 Integron Affect Expression of a Gene Cassette Possessing a Canonical Shine-Dalgarno Sequence.

By searching the Integrall integron and GenBank databases, a novel open reading frame (ORF) of 51 nucleotides (nts) (ORF-17) overlapping the previously described ORF-11 was identified within the attI1 site in virtually all class 1 integrons. Using a set of isogenic plasmid constructs carrying a single gene cassette (blaGES-1) and possessing a canonical translation initiation region, we found that ORF-17 contributes to GES-1 expression.

Carbapenemase-producing Klebsiella pneumoniae bloodstream infections: lowering mortality by antibiotic combination schemes and the role of carbapenems.

Carbapenemase-producing Klebsiella pneumoniae strains (CP-Kps) are currently among the most important nosocomial pathogens. An observational study was conducted during 2009 to 2010 in two hospitals located in a high-prevalence area (Athens, Greece). The aims were (i) to evaluate the clinical outcome of patients with CP-Kp bloodstream infections (BSIs), (ii) to identify predictors of mortality, and (iii) to evaluate the various antibiotic schemes employed. A total of 205 patients with CP-Kp BSIs were identified: 163 (79.5%) were infected with KPC or KPC and VIM, and 42 were infected with VIM producers. For definitive treatment, 103 patients received combination therapy (two or more active drugs), 72 received monotherapy (one active drug), and 12 received therapy with no active drug. The remaining 18 patients died within 48 h after the onset of bacteremia. The all-cause 28-day mortality was 40%. A significantly higher mortality rate was observed in patients treated with monotherapy than in those treated with combination therapy (44.4% versus 27.2%; P=0.018). The lowest mortality rate (19.3%) was observed in patients treated with carbapenem-containing combinations. In the Cox proportion hazards model, ultimately fatal disease (hazards ratio [HR], 3.25; 95% confidence interval [CI], 1.51 to 7.03; P=0.003), the presence of rapidly fatal underlying diseases (HR, 4.20; 95% CI, 2.19 to 8.08; P<0.001), and septic shock (HR, 2.15; 95% CI, 1.16 to 3.96; P=0.015) were independent predictors of death. Combination therapy was strongly associated with survival (HR of death for monotherapy versus combination, 2.08; 95% CI, 1.23 to 3.51; P=0.006), mostly due to the effectiveness of the carbapenem-containing regimens.

Confronting carbapenemase-producing Klebsiella pneumoniae.

The ongoing spread of carbapenemase-producing (CP) multidrug-resistant enterobacteria, primarily Klebsiella pneumoniae, has undoubtedly caused a public health crisis of unprecedented dimensions. The scientific community has been struggling with these highly problematic nosocomial pathogens for more than a decade. Faced with the current situation, one cannot help but wish we could have done better, earlier. However, significant steps have been and are currently being made towards a better understanding of transmission routes of CP microorganisms and in designing strategies that could effectively curb this devastating epidemic. Most importantly, the systematic evaluation of accumulating experimental and clinical data has paved the way to a more rational management of CP-infected patients. In addition, systematic efforts of the industry have led to the development of novel antibacterial agents that are active against CP strains and expected to be introduced to clinical practice in the immediate future.

Interactions of oximino-substituted boronic acids and ß-lactams with the CMY-2-derived extended-spectrum cephalosporinases CMY-30 and CMY-42.

CMY-30 and CMY-42 are extended-spectrum (ES) derivatives of CMY-2. ES characteristics are due to substitutions of Gly (CMY-30) and Ser (CMY-42) for Val211 in the Ω-loop. To characterize the effects of 211 substitutions, we studied the interactions of CMY-2, -30, and -42 with boronic acid transition state inhibitors (BATSIs) resembling ceftazidime and cefotaxime, assessed thermal stability of the enzymes in their free forms and in complexes with BATSIs and oximino-ß-lactams, and simulated, using molecular dynamics (MD), the CMY-42 apoenzyme and the CMY-42 complexes with ceftazidime and the ceftazidime-like BATSI. Inhibition constants showed that affinities between CMY-30 and CMY-42 and the R1 groups of BATSIs were lower than those of CMY-2. ES variants also exhibited decreased thermal stability either as apoenzymes or in covalent complexes with oximino compounds. MD simulations further supported destabilization of the ES variants. Val211Ser increased thermal factors of the Ω-loop backbone atoms, as previously observed for CMY-30. The similar effects of the two substitutions seemed to be due to a less-constrained Tyr221 likely inducing concerted movement of elements at the edges of the active site (Ω-loop-Q120 loop-R2 loop/H10 helix). This inner-protein movement, along with the wider R1 binding cleft, enabled intense vibrations of the covalently bound ceftazidime and ceftazidime-like BATSIs. Increased flexibility of the ES enzymes may assist the productive adaptation of the active site to the various geometries of the oximino substrates during the reaction (higher frequency of near-attack conformations).

Bloodstream infections caused by carbapenemase-producing Klebsiella pneumoniae: a clinical perspective.

Dissemination of carbapenemase-producing Klebsiella pneumoniae (CP-Kp) has caused a public health crisis that can be paralleled with that caused by the spread of MRSA. CP-Kps, being multidrug-resistant, mainly affect patients with severe underlying conditions in the acute-healthcare setting. CP-Kps are responsible for a variety of life-threatening infections including bacteremia and pneumonia. The shortage of therapeutic options has forced clinicians to use colistin as well as tigecycline, a novel bacteriostatic agent. Although both drugs are generally active in vitro against CP-Kps, therapeutic failures, especially in bacteremias, are quite common. The authors suggest here, after reviewing the literature, that use of the latter drugs should be re-assessed and optimized. The authors have also summarized experimental and clinical data indicating that exploitation of the pharmacokinetic/pharmacodynamic features of carbapenems may provide solutions in bloodstream infections caused by CP-Kps with low-level resistance to the latter drugs. Most importantly, there is evidence that monotherapy must be avoided.

Emergence of Escherichia coli sequence type 410 (ST410) with KPC-2 ß-lactamase.

Fifteen carbapenem-non-susceptible Escherichia coli isolates obtained during the period May 2010 to April 2011 in a hospital and a long-term care facility (LTCF) in Larissa (Central Greece) were investigated. Minimum inhibitory concentrations (MICs) to various antimicrobial agents were determined by Etest. Carriage of bla genes, including bla(KPC-2) and bla(CTX-M), was documented by polymerase chain reaction (PCR) and sequencing. Production of ß-lactamases was confirmed by isoelectric focusing. Transfer of resistance was carried out by conjugation. Plasmid incompatibility groups were determined by PCR-based replicon typing and replicon sequence typing. Isolates were genotyped by multilocus sequence typing. Ten E. coli isolates with KPC-2 were derived from seven patients in the University Hospital of Larissa. Six patients had previously been treated for prolonged time periods in a LTCF located in the same city. The remaining isolate was from a patient previously treated in an Athens hospital. Screening of faecal samples from 20 randomly selected LTCF patients yielded eight enterobacteria with KPC-2, of which five were E. coli, showing the wide spread of KPC-2-producers in this institution and confirming that it was the focus of the outbreak. Fourteen of the isolates were classified as sequence type 410 (ST410); the remaining isolate belonged to a novel ST (ST2281). All 15 isolates carried a KPC-2-encoding plasmid of the Inc group FIIK. Additional plasmids encoding enzymes of the CTX-M-1 family were identified in 11 isolates. The bla(KPC-2)-carrying plasmid IncFIIK, widespread amongst Klebsiella pneumoniae in Greece, has probably been acquired by E. coli ST410 known to be associated with CTX-M production. Diffusion of bla(KPC-2) in common pathogens such as E. coli is of concern.

Effects of the Val211Gly substitution on molecular dynamics of the CMY-2 cephalosporinase: implications on hydrolysis of expanded-spectrum cephalosporins.

CMY-30, a naturally occurring class C ß-lactamase differing from the Citrobacter freundii-derived CMY-2 by a Val211Gly substitution in the Ω-loop, exhibits increased hydrolytic efficiency against ceftazidime and cefotaxime. Kinetic constants of CMY-2 and CMY-30 against the latter substrates suggested that the improved efficiency of the Gly211 variant was due to an increase in k(cat). The structural basis of the increased turn-over rates of oxyimino-cephalosporins caused by Val211Gly was studied using 5 ns molecular dynamics simulations of CMY-2 and CMY-30 in their free forms and in covalent complexes with ceftazidime (acyl-enzyme) as well as a boronic acid analogue of ceftazidime (deacylation transition state). Analysis of thermal factors indicated that Val211Gly increased the flexibility of the Ω-loop/H7-helix and the Q120-loop formed by amino acids 112-125, and also altered the vibrations of the H10-helix/R2-loop. Structural elements containing the catalytic residues remained relatively rigid except Tyr150 in acyl-enzyme species. Regions exhibiting altered flexibility due to the substitution appear to move in a concerted manner in both enzymes. This movement was more intense in CMY-30 and also at directions different to those observed for CMY-2. Additionally, it appeared that the Val211Gly increased the available space for the accommodation of the R1 side chain of ceftazidime. These findings are likely associated with the significantly increased vibrations of the bound compounds observed in CMY-30 complexes. Therefore, the extended spectrum properties of CMY-30 seem to arise through a complex process implicating changes in protein movement and in the mode of substrate accommodation.

An update of the evolving epidemic of blaKPC-2-carrying Klebsiella pneumoniae in Greece (2009-10).

OBJECTIVES: To follow the epidemic of KPC-2-producing Klebsiella pneumoniae in Greece. METHODS: KPC-2-producing isolates (n = 378) were collected during January 2009-April 2010 in 40 Greek hospitals. bla(KPC) and bla(VIM) were detected by PCR. Carbapenemase production was confirmed by spectrophotometry. Sequences flanking bla(KPC-2) and their plasmid carriers were studied. Isolates were typed by PFGE and multilocus sequence typing (MLST). RESULTS: All 378 isolates were bla(KPC-2) positive; 18 also carried bla(VIM-1/VIM-4). Higher isolation frequencies were observed in Athens and Crete. Isolates were classified into 13 PFGE types and 11 sequence types (STs). ST258 was predominant (n = 322), followed by ST147 (n = 20), ST383 (n = 9), ST133 (n = 6), ST274 (n = 4) and ST323 (n = 3). Of the remaining isolates, seven were distributed into five STs (11, 17, 340 and the novel 494 and 495) and seven were not typed. bla(KPC-2) could not be transferred from ST258 isolates, in contrast to isolates of ST17, ST133, ST147, ST274, ST494 and ST495. All bla(KPC-2)-encoding plasmids were of similar size (∼100 kb) and showed indistinguishable restriction fragment length polymorphism (RFLP) patterns except those from the ST340 isolates. Sequences flanking bla(KPC-2) revealed that the Tn4401a isoform was present in plasmids from all STs except ST340 containing Tn4401b. Co-production of VIM enzymes was observed in isolates of ST147, ST323 and ST383. CONCLUSIONS: Apart from the epidemic of KPC-2-producing K. pneumoniae belonging to ST258 in Greece, diffusion of bla(KPC-2) to at least 10 additional STs has taken place. Notably, strains from three of the latter STs (147, 323 and 383) were found to carry both bla(KPC-2) and bla(VIM).

Comparative biochemical and computational study of the role of naturally occurring mutations at Ambler positions 104 and 170 in GES ß-lactamases.

In GES-type ß-lactamases, positions 104 and 170 are occupied by Glu or Lys and by Gly, Asn, or Ser, respectively. Previous studies have indicated an important role of these amino acids in the interaction with ß-lactams, although their precise role, especially that of residue 104, remains uncertain. In this study, we constructed GES-1 (Glu104, Gly170), GES-2 (Glu104, Asn170), GES-5 (Glu104, Ser170), GES-6 (Lys104, Ser170), GES-7 (Lys104, Gly170), and GES-13 (Lys104, Asn170) by site-specific mutagenesis and compared their hydrolytic properties. Isogenic comparisons of ß-lactam resistance levels conferred by these GES variants were also performed. Data indicated the following patterns: (i) Lys104-containing enzymes exhibited enhanced hydrolysis of oxyimino-cephalosporins and reduced efficiency against imipenem in relation to enzymes possessing Glu104, (ii) Asn170-containing enzymes showed reduced hydrolysis rates of penicillins and older cephalosporins, (iii) Ser170 enabled GES to hydrolyze cefoxitin efficiently, and (iv) Asn170 and Ser170 increased the carbapenemase character of GES enzymes but reduced their activity against ceftazidime. Molecular dynamic simulations of GES apoenzyme models, as well as construction of GES structures complexed with cefoxitin and an achiral ceftazidime-like boronic acid, provided insights into the catalytic behavior of the studied mutants. There were indications that an increased stability of the hydrogen bonding network of Glu166-Lys73-Ser70 and an altered positioning of Trp105 correlated with the substrate spectra, especially with acylation of GES by imipenem. Furthermore, likely effects of Ser170 on GES interactions with cefoxitin and of Lys104 on interactions with oxyimino-cephalosporins were revealed. Overall, the data unveiled the importance of residues 104 and 170 in the function of GES enzymes.

Risk factors for bloodstream infection with Klebsiella pneumoniae producing VIM-1 metallo-beta-lactamase.

OBJECTIVES: To identify risk factors for bloodstream infections (BSIs) caused by VIM-1-producing Klebsiella pneumoniae (VPKP). METHODS: Consecutive patients with K. pneumoniae BSIs were identified in three tertiary care hospitals between February 2004 and March 2006. Patients infected with VPKP were designated as cases and those infected with non-VPKP as controls. Potential risk factors for VPKP BSIs were examined by univariate and multivariate analysis. RESULTS: A total of 178 patients with K. pneumoniae BSIs were identified; 67 (37.6%) were infected with VPKP (cases) and 111 with non-VPKP (controls). In multivariate analysis, cases were more likely to have been in an intensive care unit (ICU) [odds ratio (OR), 6.78; 95% confidence interval (CI), 2.69-17.06; P < 0.001], have had prior exposure to >3 different classes of antibiotics (OR, 12.6; 95% CI, 2.17-73.27; P = 0.01) and have had prior use of carbapenems (OR, 2.83; 95% CI, 1.07-7.49; P = 0.03). CONCLUSIONS: Stay in an ICU, prior use of carbapenems and prior exposure to >3 different classes of antibiotics were independent predictors for VPKP BSIs. These findings provide guidance for antibiotic policies and infection control strategies to contain the spread of VPKP.