oprD Mutations Research Articles

Antibacterial effect of imipenem/relebactam on aerobic Gram-negative bacilli: in vitro simulations of 7 or 14 day human exposures.

We assessed the antibacterial effect of human simulations of dosing with imipenem/relebactam (with or without amikacin) on Enterobacteriaceae or Pseudomonas aeruginosa over 7 or 14 day antibiotic exposures. An in vitro pharmacokinetic model was used to assess changes in bacterial load and population profiles. Imipenem/relebactam produced an initial >4 log drop in viable counts followed by suppression for 7 days for Enterobacteriaceae whether the strain was WT, produced KPC enzymes or produced an AmpC enzyme with porin loss. Similarly, with the P. aeruginosa strains, there was an initial >4 log clearance over the first 24 h irrespective of whether the strain was WT, hyperexpressed AmpC or had OprD mutation with porin loss. However, with three of four strains there was modest regrowth over the 7 days. There were no changes in imipenem/relebactam MICs over the 7 days. Addition of amikacin in 7 day simulations resulted in more suppression of pseudomonal growth. In 14 day simulations with P. aeruginosa there was regrowth to 8 log10 by 14 days with imipenem/relebactam alone and associated increases in MICs. Addition of amikacin resulted in clearance from the model and prevented changes in population profiles. Imipenem/relebactam was highly effective at reducing the bacterial load of Enterobacteriaceae and there was no emergence of resistance. Against P. aeruginosa, the initial bacterial burden was also rapidly reduced, but there was subsequent regrowth, especially after 7 days of exposure. Addition of amikacin increased the clearance of P. aeruginosa and prevented emergence of resistance.

First detection of insertion sequences ISpa1635 and IS1411 among non-carbapenemase producing strains of Pseudomonas aeruginosa in Kerman, Iran

BackgroundLoss of OprD, AmpC overproduction and efflux pump over activity are the main mechanism for chromosomally resistance to carbapenems in Pseudomonas aeruginosa. In this study, we investigated the chromosomally resistance mechanism to carbapenems among 9 non-carbapenemase-producing P. aeruginosa (NCPPa). Material and methodAntibacterial resistance was determined by disc diffusion method among 170 clinical isolates of P. aeruginosa. Sixty-one isolates were multiple drug resistance and resistance to imipenem, and 9 isolates (5.3%) were negative for ten carbapenemase genes and were recognized as NCPPa. To determine the mechanism of resistance in NCPPa isolates AmpC and efflux pump overproduction were evaluated. Overexpression of MexAB-oprM efflux pump system was evaluated by qReal-time PCR. Also, oprD gene sequenced to detect any insertional and point mutations. ResultsAll NCPPa isolates shown a significant overexpression of mexA gene in qReal-time PCR experiment, while 88.8% of the isolates had overproduced AmpC enzyme. Finally, oprD gene was destructed by two insertion sequences (ISs), including IS1411 and ISpa1635. ConclusionAccording to the results of this study, overexpression of efflux pumps, oprD mutation and AmpC overproduction are the main chromosomal mechanism of resistance of P. aeruginosa to carbapenems in NCPPa isolates in this study.

703. Mechanisms of High-Level Ceftolozane/Tazobactam (CT) Resistance Against Pseudomonas aeruginosa and Synergy Between CT and Tobramycin (TOB)

BackgroundCeftolozane/tazobactam (CT) is a cephalosporin/β-lactamase inhibitor with excellent activity against multi-drug-resistant (MDR) P. aeruginosa (PSA). Several cases of CT-resistance (CT-R) development after exposure have been reported. We recovered a PSA isolate with high-level CT-R from a bacteremic patient with severe, prolonged neutropenia and 5 weeks of CT exposure. Then, multiple mutational pathways and the role of combination therapy were evaluated.MethodsMinimum inhibitory concentrations (MIC) to CT were determined by Etest. Synergy tests between CT and tobramycin (TOB) were conducted and interpreted based on the fractional inhibitory concentration index (FICI). Furthermore, whole genome sequencing was performed. Paired-end reads were mapped and compared with reference strain PAO1. Variant analyses were conducted using CLC Genomics Workbench.ResultsThe MICs for CT and TOB were >256 and 4 mg/L, respectively. The combination revealed synergistic effects (FICI < 0.5) with reduced CT and TOB MICs to 16 and 1 mg/L, respectively. Clinically, combination therapy of CT 3g q8h given over 4 hours and TOB 7 mg/kg q24h successfully cleared the bacteremia within 2 days. Genomic analysis revealed the CT-R isolate contained multiple variants in the ampC gene, including G183D associated with low level CT-R. Two additional variants in aminoacid position 79 (R79Q) and position 105 (T105A) were located inside or near helix-H2 which interacts with the Ω-loop through hydrogen-binding rendering the serine active site more pliable to accommodate larger molecules. Moreover, the CT-R isolate showed a truncated ampD and multiple mutations in mexD, mexT, mexI, and mexR, a primary regulator of mexAB-oprM. The isolate also contained the oprD mutation (Q142X) and an oprD-inactivating mutation (W417X). In addition to these chromosomal mutations, the isolate harbored OXA-50, blaPAO1, and aph(3′)-IIb.ConclusionHigh-level CT-R was likely driven by multiple mutations in the ampC region causing structural changes. While resistance to CT is worrisome, our case of a severe neutropenic patient who rapidly cleared bacteremia on CT 3g qh8 given over 4 hours plus TOB emphasizes the importance of strategic combination and dosing in combating MDR PSA.Disclosures All authors: No reported disclosures.

Molecular characterization of carbapenem-resistant Gram-negative bacilli clinical isolates in Algeria.

ObjectivesThe aims of this study were to investigate the occurrence of carbapenem-resistant Gram-negative bacilli (GNB) isolated from inpatients and outpatients in Algeria between July and September 2015, and to screen their resistance mechanisms and genetic relatedness.Materials and methodsA total of 68 non-redundant isolates were identified using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) antibiotic susceptibility testing was performed using disk diffusion and Etest methods. Carbapenemase activity was carried out using modified Carba NP test, EDTA assay, and the modified Hodge test. Molecular characterization of carbapenemases and extended-spectrum β-lactamase (ESBL) genes were detected by standard PCR and sequencing. Genotyping of carbapenem-resistant isolates was performed by multilocus sequence typing (MLST) analysis.ResultsOf the 68 GNB isolates, 13 (19%) showed reduced susceptibility to carbapenems, including, four Klebsiella pneumoniae, one Escherichia coli, six Acinetobacter baumannii, and two Pseudomonas aeruginosa. blaOXA-48 gene was detected in the five Enterobacteriaceae isolates, and blaOXA-23 was identified in all A. baumannii isolates. OprD mutations were revealed in the two P. aeruginosa isolates. A total of 11 out of the 13 carbapenem-resistant GNB were detected in inpatients, and the two remaining strains were isolated from outpatients. Molecular typing showed the presence of four sequence types (STs) among the OXA-48-producing K. pneumoniae isolates: ST101, ST147, ST163, and ST2017. ST533 was identified for the OXA-48 producing E. coli isolate. All of the A. baumannii and P. aeruginosa were assigned to the international clonal lineages ST2 and ST654, respectively.ConclusionThis study reports the first detection of the epidemic multidrug-resistant lineage, K. pneumoniae ST147 coproduced blaOXA-48 and ESBL genes in Algeria and represents the first description of OXA-48-producing E. coli ST533 and K. pneumoniae ST163 and ST2017. In addition, this study describes for the first time the emergence of OXA-48-producing E. coli and K. pneumoniae in the community in Algeria, leading to major problems for managing microbial infections.

Mechanisms leading to in vivo ceftolozane/tazobactam resistance development during the treatment of infections caused by MDR Pseudomonas aeruginosa.

Characterization of the mechanisms driving ceftolozane/tazobactam resistance development in 5 of 47 (10.6%) patients treated for MDR Pseudomonas aeruginosa infections in a Spanish hospital. Five pairs of ceftolozane/tazobactam-susceptible/resistant P. aeruginosa isolates were studied. MICs were determined by broth microdilution, clonal relatedness was assessed by MLST and resistance mechanisms were investigated by phenotypic and genotypic methods, including WGS. ampC variants were cloned to assess their impact on resistance. In all five cases, the same clone was detected for the susceptible/resistant pairs; the widespread ST175 high-risk clone in four of the cases and ST179 in the remaining case. Genomic analysis of the four initial ST175 isolates revealed the characteristic OprD mutation (Q142X) responsible for carbapenem resistance and the AmpR mutation (G154R) responsible for AmpC overexpression and β-lactam resistance. The final isolates had developed ceftolozane/tazobactam and ceftazidime/avibactam resistance, and each additionally showed a mutation in AmpC: E247K in one of the isolates, T96I in two isolates and a deletion of 19 amino acids (G229-E247) in the remaining isolate. The cloned AmpC variants showed greatly increased ceftolozane/tazobactam and ceftazidime/avibactam MICs compared with WT AmpC, but, in contrast, yielded lower MICs of imipenem, cefepime and particularly piperacillin/tazobactam. On the other hand, ceftolozane/tazobactam resistance development in ST179 was shown to be driven by the emergence of the extended-spectrum OXA β-lactamase OXA-14, through the selection of an N146S mutation from OXA-10. Modification of intrinsic (AmpC) and horizontally acquired β-lactamases appears to be the main mechanism leading to ceftolozane/tazobactam resistance in MDR P. aeruginosa.

High-level resistance to meropenem in clinical isolates of Pseudomonas aeruginosa in the absence of carbapenemases: role of active efflux and porin alterations

High-level carbapenem resistance is worryingly increasing in clinical isolates and is often attributed to carbapenemase expression. This study aimed to determine the mechanisms leading to high-level meropenem resistance in six carbapenemase-negative Pseudomonas aeruginosa isolated from cystic fibrosis (CF) patients and seven carbapenemase-positive isolates from patients suffering from hospital-acquired pneumonia (HAP). MICs were determined in the absence or presence of l-arginine or glycine–glutamate as competitive substrates for OprD (OccD1) or OpdP (OccD3), respectively, or the efflux pump inhibitor Phe-Arg β-naphthylamide (PAβN). β-Lactamases were screened by phenotypic tests and/or PCR. The oprD gene and its promoter were sequenced; protein expression was evidenced by SDS-PAGE. mexA, mexX, mexC and mexE transcripts were evaluated by real-time and semiquantitative PCR. Meropenem/imipenem MICs were 64–128/16–32 mg/L and 128/128–256 mg/L in CF and HAP isolates, respectively; PAβN reduced meropenem MICs to 4–16 mg/L only and specifically in CF isolates; porin competitors had no effect on MICs. All isolates showed an increase in transcription levels of mexA, mexX and/or mexC and mutations in oprD leading to production of truncated proteins. AmpC-type cephalosporinases were overexpressed in CF isolates and VIM-2 was expressed in HAP isolates. Antibiotic exclusion from bacteria by concomitant efflux and reduced uptake is sufficient to confer high-level resistance to meropenem in isolates overexpressing AmpC-type cephalosporinases. As efflux is preponderant in these isolates, it confers a paradoxical phenotype where meropenem is less active than imipenem. Concomitant susceptibility testing of both carbapenems and rapid elucidation of the most probable resistance mechanisms is thus warranted.

First Detection of VIM-4-Producing Pseudomonas aeruginosa and OXA-48-Producing Klebsiella pneumoniae in Northeastern (Annaba, Skikda) Algeria.

The aim of the study was to investigate the prevalence and molecular support of carbapenem resistance in gram-negative bacilli clinical isolates collected between March 2013 and March 2015 in three cities (Annaba, Skikda, and Guelma) in northeastern Algeria. One hundred eighty-six isolates were identified as Enterobacteriaceae (161), Pseudomonas aeruginosa (18), and Acinetobacter baumannii (7). Thirty-six of 186 (19.3%) were resistant to carbapenems. Among them, 11 harbored carbapenemase genes, including blaOXA-48 (2 Klebsiella pneumoniae), blaVIM-4 (2 P. aeruginosa), blaNDM-1 (2 A. baumannii), and blaOXA-23 (5 A. baumannii). In addition, other β-lactamases were detected: blaCTX-M-(15/66/139), blaSHV-(28/85/1/133), and blaTEM-1. All imipenem-resistant P. aeruginosa displayed OprD mutations. Multilocus sequence typing demonstrated the presence of ST 404 and ST 219 in K. pneumoniae, ST 2 and ST 85 in A. baumannii, and ST (244, 1076, 241, 227, and 233) in P. aeruginosa. In this study, we report the first detection of P. aeruginosa ST 1076 harboring the blaVIM-4 gene in African countries in two cities (Annaba and Skikda) in northeastern Algeria. Additionally, we report the first detection of blaOXA-48 in K. pneumoniae ST 404 and ST 219 in Algerian cities (Annaba and Skikda).

Overproduction of active efflux pump and variations of OprD dominate in imipenem-resistant Pseudomonas aeruginosa isolated from patients with bloodstream infections in Taiwan.

BackgroundThe emergence of imipenem-resistant Pseudomonas aeruginosa (IRPA) has become a great concern worldwide. The aim of this study was to investigate resistance mechanisms associated with bloodstream isolated IRPA strains in Taiwan.ResultsA total of 78 non-duplicated IRPA isolates were isolated from patients with bloodstream infection. The average prevalence of imipenem-resistance in those isolates was 5.9 % during a 10-year longitudinal surveillance in Taiwan. PFGE results showed high clonal diversity among the 78 isolates. VIM-2, VIM-3, OXA-10, and OXA-17 β-lactamases were identified in 2 (2.6 %), 3 (3.8 %), 2 (2.6 %), and 1 (1.3 %) isolates, respectively. Active efflux pumps, AmpC β-lactamase overproduction, and extended-spectrum AmpC cephalosporinases (ESACs) were found in 58 (74.4 %), 25 (32.1 %) and 15 (19.2 %) of IRPA isolates, respectively. oprD mutations with amino acid substitution, shortened putative loop L7, premature stop codon caused by point mutation, frameshift by nucleotide insertion or deletion, and interruption by insertion sequence were found in 19 (24.4 %), 18 (23.1 %), 15 (19.2 %), 14 (17.9 %), and 10 (12.8 %) of isolates, respectively.ConclusionsThis study suggests that alterations in the OprD protein and having an active efflux pump are the main mechanisms associated with bloodstream isolated IRPA. Overproduction of AmpC, ESACs, and the presence of VIM- and OXA-type β-lactamases play additional roles in reduced susceptibility to imipenem in P. aeruginosa isolates in Taiwan.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0719-2) contains supplementary material, which is available to authorized users.

Emergence of Storm Resistant Mechanisms in Pseudomonas aeruginosa Isolated from Burn Patients Hospitalized in Ghotbeddin Shirazi Burn Hospital

Introduction: Pseudomonas aeruginosa is a bacterium has capability of presenting different policies against antimicrobial agents. These capacities make this bacterium as one of the most important cause of nosocomial infection especially among burned patients. Methods: In this cross sectional study, total of 253 hospitalized burned patients (During September 2013 to November 2014) in Ghotbeddin Shirazi were evaluated for P. aeruginosa. Confirmed isolates tested for Metallo-β-lactamase (MBLs) producing and related genes bla IMPI, II , bla SPM , bla KHM , bla VIM , efflux pump activity, and OprD mutations. Results: From total of 56 (22%) P. aeruginosa isolates, 32 (57.14%) were MBLs while in genetic evaluation, only bla IMPII, bla KHM and bla VIM genes were detected. While 52 (92.8%) isolates had MexAB-OprM gene but 44(78.6%) isolates had efflux pump activity. In OprD sequencing evaluation it has been proved that the most imipenem resistance isolates have mutations in this related gene. Based on the results it has been proved that the circulating P. aeruginosa isolates in Shiraz burn center are armed with the most potential resistant mechanisms. Conclusion: According to that the most isolates have the potential of express different mechanisms of resistance, it is highly recommended to evaluate the circulating infectious agents periodically and determine their resistant algorithm to control them before being resistant and being dominant in health centers.

Exposure to Reactive Oxygen Species and Piperacillin Leads to Multidrug Resistance in Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa is one of the most common causes of nosocomial infections. Hospital associated infection by multidrug resistant (MDR) strains constitute a serious problem worldwide. The aim of the present study was to evaluate whether exposure to sub-MIC levels of anti-pseudomonas antibiotics and reactive oxygen species (ROS), such as hydroxyl radicals, could lead to MDR of P. aeruginosa. P. aeruginosa standard strain PAO1 was used in this study. All five anti-pseudomonas agents, that is, piperacillin, levofloxacin, meropenem, ceftazidime and amikacin, were investigated for induction of resistance and cross-resistance in vitro. The reference strain was incubated 24 h and transferred 5 times after being exposed to 1 mM H2O2 in addition to a sub-MIC of each antibiotic by the agar dilution method. When cross-resistance to another antibiotic was confirmed, ampC, mexAB, and oprD expression and mutation of QRDR were investigated. Sub-MIC of piperacillin induced resistance to piperacillin and levofloxacin under stimulation with ROS. The mechanism of multi-resistance to β-lactams and levofloxacin was confirmed by RT-PCR. It was a decrease of oprD expression (p<0.05). The increase of MIC was inhibited by the ROS scavenger sodium zinc histidine dihydrolipoyl histidinate (DHL-His-Zn). In conclusion, for P. aeruginosa PAO1 to acquire multidrug resistance, stimulation with ROS was as important as exposure to sub-MIC of piperacillin.

In vivo evolution of resistance of Pseudomonas aeruginosa strains isolated from patients admitted to an intensive care unit: mechanisms of resistance and antimicrobial exposure.

The main objective of this study was to investigate the relationship among the in vivo acquisition of antimicrobial resistance in Pseudomonas aeruginosa clinical isolates, the underlying molecular mechanisms and previous exposure to antipseudomonal agents. PFGE was used to study the molecular relatedness of the strains. The MICs of ceftazidime, cefepime, piperacillin/tazobactam, imipenem, meropenem, ciprofloxacin and amikacin were determined. Outer membrane protein profiles were assessed to study OprD expression. RT-PCR was performed to analyse ampC, mexB, mexD, mexF and mexY expression. The presence of mutations was analysed through DNA sequencing. We collected 17 clonally related paired isolates [including first positive samples (A) and those with MICs increased ≥4-fold (B)]. Most B isolates with increased MICs of imipenem, meropenem and ceftazidime became resistant to these drugs. The most prevalent resistance mechanisms detected were OprD loss (65%), mexB overexpression (53%), ampC derepression (29%), quinolone target gene mutations (24%) and increased mexY expression (24%). Five (29%) B isolates developed multidrug resistance. Meropenem was the most frequently (71%) received treatment, explaining the high prevalence of oprD mutations and likely mexB overexpression. Previous exposure to ceftazidime showed a higher impact on selection of increased MICs than previous exposure to piperacillin/tazobactam. Stepwise acquisition of resistance has a critical impact on the resistance phenotypes of P. aeruginosa, leading to a complex scenario for finding effective antimicrobial regimens. In the clinical setting, meropenem seems to be the most frequent driver of multidrug resistance development, while piperacillin/tazobactam, in contrast to ceftazidime, seems to be the β-lactam least associated with the selection of resistance mechanisms.

Emergence of Carbapenem-Resistant Pseudomonas aeruginosa and Acinetobacter baumannii Clinical Isolates Collected from Some Libyan Hospitals.

The aim of the present study was to investigate the molecular mechanism of carbapenem resistance in Pseudomonas aeruginosa and Acinetobacter baumannii clinical isolates recovered from Libyan hospitals between April 2013 and April 2014. In total, 49 strains (24 P. aeruginosa and 25 A. baumannii) were isolated, including 21 P. aeruginosa and 22 A. baumannii isolates (87.75%) resistant to imipenem (minimum inhibitory concentrations ≥16 μg/ml). The blaVIM-2 gene was detected in 19 P. aeruginosa isolates. All imipenem-resistant P. aeruginosa isolates showed the presence of OprD mutations. Acquired OXA-carbapenemase-encoding genes were present in all A. baumannii isolates: blaOXA-23 (n=19) and blaOXA-24 (n=3). Finally, a total of 13 and 17 different sequence types were assigned to the 21 P. aeruginosa and the 22 A. baumannii carbapenem-resistant isolates, respectively. This study is the first report describing imipenem-resistant P. aeruginosa and A. baumannii isolated from patients in Libya. We report the first case of co-occurrence of blaVIM-2 with oprD porin loss in identical isolates of P. aeruginosa in Libya and demonstrate that these oprD mutations can be used as a tool to study the clonality in P. aeruginosa isolates. We also report the first identification of multidrug-resistant A. baumannii isolates harboring blaOXA-23-like, blaOXA-24-like, and blaOXA-48-like genes in Libya.

Detection of Pseudomonas aeruginosa isolates carrying the bla OXA-142 extended-spectrum β-lactamase gene in Taiwan

The emergence of extended-spectrum β-lactamase (ESBL) OXA-142 gene (blaOXA-142) in Pseudomonas aeruginosa has been reported in Bulgaria and other European countries, but rarely in Asia. From January 2009 to December 2012, 90 P. aeruginosa isolates with reduced susceptibility or resistance to ceftazidime (minimum inhibitory concentration≥8mg/L) were screened for ESBL and other broad-spectrum β-lactamase genes by polymerase chain reaction and sequencing. Clonal relationship of the isolates was determined by pulsed-field gel electrophoresis. Three isolates were positive for ESBL production, exhibited resistance to ceftazidime, and carried the blaOXA-142 gene. The blaOXA-142 gene was integrated into class 1 integron. Using Southern blot analysis, blaOXA-142-containing integron was found to be located on a plasmid in all three isolates. Eleven strains of P. aeruginosa carrying blaOXA-17 gene were found. The oprD mutation was found in all the 21 ESBL strains of P. aeruginosa. This study confirmed the presence of blaOXA-142-positive P. aeruginosa isolates in Taiwan.

Reduced production of OprM may promote oprD mutations and lead to imipenem resistance in Pseudomonas aeruginosa carrying an oprD-group 1A allele.

Resistance mechanisms in a group of carbapenemase-negative Pseudomonas aeruginosa that were susceptible to all antibiotics except carbapenems (carbapenem resistance-only P. aeruginosa [CROPA]) were studied. Ten genetically nonrelated CROPA isolates and their carbapenem-susceptible counterparts were further investigated. OprD production was demonstrated by protein electrophoresis in only 1 of the 10 carbapenem-susceptible isolates, while the other 9 isolates showed hyperproduction of OprM. DNA sequencing of oprD revealed a shortened loop 7 domain (group 1A allele) in eight carbapenem-susceptible isolates. Various oprD mutations, leading to early terminations, were found in 9 of the 10 CROPA isolates. RNA analysis demonstrated hyperexpression of oprM with normal expression of mexA in eight of the carbapenem-susceptible isolates, while in seven of their CROPA counterparts, the oprM expression was significantly reduced. Deletion of oprM was performed in two pairs of representative isolates. Selection of imipenem resistant variants by a disc assay indicated that the lost-of-function mutations in oprD occurred relatively faster in the ΔoprM mutants compared with their corresponding parent strains. Under selection pressure, reduced production of OprM may promote the selection of spontaneous changes in oprD, resulting in the carbapenem resistance in a group of pan-susceptible P. aeruginosa isolates characterized by harboring an oprD-group 1A allele.

Carbapenem-resistant-only Pseudomonas aeruginosa infection in patients formerly infected by carbapenem-susceptible strains

Pseudomonas aeruginosa isolates that were initially carbapenem-susceptible and later became selective carbapenem-resistant following antimicrobial therapy were identified from individual cases during the same hospitalisation. Cross-resistance to other β-lactams was not found and their susceptibilities remained identical in consecutive isolates. Real-time quantitative reverse transcription PCR was performed to investigate the role of OprD, an outer membrane protein regulating the entry of carbapenems, in the appearance of carbapenem-resistant-only P. aeruginosa (CROPA). Fifteen paired isolates of carbapenem-susceptible P. aeruginosa (CS-PA) and CROPA were identified. All of the cases had carbapenem exposure history within 1 month before the appearance of CROPA (mean 10 days). Reduced OprD expression was found in 93% (14/15) of the isolates, suggesting that oprD inactivation was the major contributor to selective carbapenem resistance. Of the 14 cases with CROPA due to oprD mutation, 71% (10/14) were persistent infection, as genotype analysis revealed that their paired strains were isogenic; 29% (4/14) represented re-infections as they were heterogenic, suggesting that oprD-deficient CROPA existed in the hospital and that carbapenem selective pressure promoted its spread to patients. We conclude that CROPA may occur soon after the use of carbapenems to treat CS-PA infections and that oprD mutation is the major mechanism of resistance in CROPA. Restriction of empirical use of carbapenems by antibiotic stewardship is important to halt the occurrence of CROPA.

Molecular Epidemiology of Carbapenem-Resistant Pseudomonas aeruginosa Clinical Strains Isolated from Western Algeria Between 2009 and 2012

Infections caused by carbapenem-resistant Pseudomonas aeruginosa strains represent a major therapeutic and epidemiological problem. The aim of this study was to characterize carbapenem resistance in 89 clinical strains of P. aeruginosa isolated from three hospitals in western Algeria between October 2009 and November 2012. Minimum inhibitory concentrations (MICs) of imipenem were determined by the Etest method. Screening for metallo-β-lactamase (MβL) was performed using Etest MβL strips, and a PCR was conducted to detect carbapenemase-encoding genes. The amplification of the oprD gene followed by a sequencing reaction was performed for all strains resistant to imipenem. The clonality of 53 P. aeruginosa strains was demonstrated using multilocus sequence typing (MLST). Among the 89 isolates, 35 (39.33%) were found to be resistant to IMP (MICs ≥16 μg/ml). The blaVIM-2 gene was detected in two strains. The remaining imipenem-resistant isolates showed the presence of oprD mutations. The MLST analysis differentiated strains into various clones and the strains from the same clone had an identical sequence of the oprD gene. We report the second detection in 2010 of blaVIM-2 in Algerian P. aeruginosa strains. We also found that oprD mutations were the major determinant of high-level imipenem resistance. We demonstrate that these oprD mutations can be used as a tool to study the clonality in P. aeruginosa isolates.

OprD mutations and inactivation in imipenem-resistant Pseudomonas aeruginosa isolates from China

To investigate the mechanisms involved in imipenem resistance of Pseudomonas aeruginosa in southern China, 61 imipenem-resistant P. aeruginosa clinical isolates were collected from 4 hospitals between October 2011 and June 2012. All isolates were resistant to imipenem, whereas 21.3% were susceptible or intermediate to meropenem. Variable degrees of resistance to other β-lactam and non-β-lactam antimicrobials were observed. PFGE revealed high-level of clonal diversity. Among the 61 isolates, 50 isolates had OprD loss by disrupted oprD mutations, including 43 with frameshift mutations of oprD and 7 with a premature stop codon by single point mutation. Six isolates were oprD-negative by PCR, suggestive of a major disruption of oprD genes. Five isolates had intact oprD but had reduced expression of oprD genes. In addition, only one isolate with disrupted oprD mutation by a premature stop codon was confirmed to be a metallo-β-lactamase producer (IMP-9). Our results show that the loss of OprD, as well as reduced expression of oprD and MBL production, were the predominant mechanisms of imipenem resistance in P. aeruginosa in southern China.

Analysis of the influence of drug resistance factors on the efficacy of combinations of antibiotics for multidrug-resistant Pseudomonas aeruginosa isolated from hospitals located in the suburbs of Kanto area, Japan.

Infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa are very difficult to treat. The aim of this study was to develop more effective treatments by investigating in vitro the effects of combinations of antibiotics against 47 MDR P. aeruginosa isolates harbouring various resistance factors. The isolates included 41 (87%) metallo-β-lactamase (MBL)-positive strains, 37 (79%) strains with mutations in OprD and 46 (98%) strains carrying the genes encoding aminoglycoside-modifying enzymes (AMEs). The quinolone resistance-determining region was mutated in all of the strains. These strains were classified into 16 groups according to amplified fragment length polymorphism and resistance factors. The effects of combinations of antibiotics on 16 representative strains were determined using a 'Break-point Checkerboard Plate' assay. Combinations of amikacin+aztreonam (coverage rate, 81.3%) and arbekacin+aztreonam (93.8%) inhibited growth. In contrast, combinations of ciprofloxacin+meropenem (6.3%) and ciprofloxacin+ceftazidime (12.5%) were much less effective. Aztreonam and arbekacin (or amikacin) are not substrates for MBLs and AMEs, respectively. We conclude that the combined effects of these drugs were possibly because of resistance factors.

Selective advantages of two major clones of carbapenem-resistant Pseudomonas aeruginosa isolates (CC235 and CC641) from Korea: antimicrobial resistance, virulence and biofilm-forming activity

The characteristics of carbapenem-resistant P. aeruginosa (CRPA) isolates from Korea were investigated. Two major clones, clonal complex (CC) 235 and CC641, were identified. CC235, an important international clone, might have been imported recently in Korea as this clone displayed a homogeneous genotype, oprD mutation and antimicrobial resistance profile. While 13 ST235 isolates harboured the blaIMP-6 gene, which conferred high-level meropenem resistance, CC641 isolates showed high biofilm-forming activity. CC235 and CC641 isolates showed distinct distribution of ferripyoverdine receptor type and virulence markers. While all CC235 isolates were of the fpvAIIb type and exoS(-)/exoU(+), CC641 isolates were exoS(+)/exoU(-), and all but one showed the fpvAIII type. CC235 and CC641 isolates were also characterized by different extracellular protease activity: staphylolysin and elastase activities in CC235 and CC641, respectively. Two major CRPA clones in Korea seem to be predominant, reflecting their selective advantage by virtue of antimicrobial resistance, virulence and biofilm-forming activity.

Alterations of OprD in Carbapenem-Intermediate and -Susceptible Strains of Pseudomonas aeruginosa Isolated from Patients with Bacteremia in a Spanish Multicenter Study

We investigated the presence of OprD mutations in 60 strains of metallo-ß-lactamase-negative Pseudomonas aeruginosa intermediately susceptible (IS [n = 12]; MIC = 8 μg/ml) or susceptible (S [n = 48]; MICs ≤ 1 to 4 μg/ml) to imipenem and/or meropenem that were isolated from patients with bacteremia in order to evaluate their impact on carbapenem susceptibility profiles. The presence of mutations in oprD was detected by sequencing analysis. OprD expression was assessed by both outer membrane protein (OMP) analysis and real-time PCR (RT-PCR). Fourteen (23%) isolates had an OprD identical to that of PAO1, and OprD modifications were detected in 46 isolates (77%). Isolates were classified as OprD "full-length types" (T1 [n = 40, including both wild-type OprD and variants showing several polymorphisms]) and OprD "deficient types" (T2 [n = 3 for OprD frameshift mutations] and T3 [n = 17 for premature stop codons in oprD]). RT-PCR showed that 5 OprD type T1 isolates presented reduced transcription of oprD (0.1- to 0.4-fold compared to PAO1), while oprD levels increased more than 2-fold over that seen with PAO1 in 4 OprD type T1 isolates. A total of 50% of the isolates belonging to OprD "deficient types" were susceptible to both carbapenems, and 40% were susceptible to meropenem and intermediately susceptible to imipenem. Only one isolate (5%) within this group was intermediately susceptible to both carbapenems, and one (5%) was susceptible to imipenem and intermediately susceptible to meropenem. We concluded that OprD inactivating mutations in clinical isolates of P. aeruginosa are not restricted only to carbapenem-resistant isolates but are also found in isolates with imipenem or meropenem MICs of only 0.06 to 4 μg/ml.