Role In Carbapenem Resistance Research Articles

Exploring the resistome, virulome, and mobilome of multidrug-resistant Klebsiella pneumoniae isolates: deciphering the molecular basis of carbapenem resistance

BackgroundKlebsiella pneumoniae, a notorious pathogen for causing nosocomial infections has become a major cause of neonatal septicemia, leading to high morbidity and mortality worldwide. This opportunistic bacterium has become highly resistant to antibiotics due to the widespread acquisition of genes encoding a variety of enzymes such as extended-spectrum beta-lactamases (ESBLs) and carbapenemases. We collected Klebsiella pneumoniae isolates from a local tertiary care hospital from February 2019–February 2021. To gain molecular insight into the resistome, virulome, and genetic environment of significant genes of multidrug-resistant K. pneumoniae isolates, we performed the short-read whole-genome sequencing of 10 K. pneumoniae isolates recovered from adult patients, neonates, and hospital tap water samples.ResultsThe draft genomes of the isolates varied in size, ranging from 5.48 to 5.96 Mbp suggesting the genome plasticity of this pathogen. Various genes conferring resistance to different classes of antibiotics e.g., aminoglycosides, quinolones, sulfonamides, tetracycline, and trimethoprim were identified in all sequenced isolates. The highest resistance was observed towards carbapenems, which has been putatively linked to the presence of both class B and class D carbapenemases, blaNDM, and blaOXA, respectively. Moreover, the biocide resistance gene qacEdelta1 was found in 6/10 of the sequenced strains. The sequenced isolates exhibited a broad range of sequence types and capsular types. The significant antibiotic resistance genes (ARGs) were bracketed by a variety of mobile genetic elements (MGEs). Various spontaneous mutations in genes other than the acquired antibiotic-resistance genes were observed, which play an indirect role in making these bugs resistant to antibiotics. Loss or deficiency of outer membrane porins, combined with ESBL production, played a significant role in carbapenem resistance in our sequenced isolates. Phylogenetic analysis revealed that the study isolates exhibited evolutionary relationships with strains from China, India, and the USA suggesting a shared evolutionary history and potential dissemination of similar genes amongst the isolates of different origins.ConclusionsThis study provides valuable insight into the presence of multiple mechanisms of carbapenem resistance in K. pneumoniae strains including the acquisition of multiple antibiotic-resistance genes through mobile genetic elements. Identification of rich mobilome yielded insightful information regarding the crucial role of insertion sequences, transposons, and integrons in shaping the genome of bacteria for the transmission of various resistance-associated genes. Multi-drug resistant isolates that had the fewest resistance genes exhibited a significant number of mutations. K. pneumoniae isolate from water source displayed comparable antibiotic resistance determinants to clinical isolates and the highest number of virulence-associated genes suggesting the possible interplay of ARGs amongst bacteria from different sources.

Prevalence and Molecular Mechanisms of Carbapenem Resistance among Gram-Negative Bacilli in Three Hospitals of Northern Lebanon

Carbapenem resistance (CR) is an emerging health issue. Epidemiological surveys on carbapenem-resistant Gram-negative bacilli (CR-GNB) in Lebanon remain scarce. In this study, we determined the prevalence of CR-GNB isolated between 2015 to 2019 in three hospitals in northern Lebanon: 311 CR-Enterobacterales (out of 11210; 2.8%), 155 CR-Pseudomonas (out of 1034; 15%) and 106 CR- Acinetobacter (out of 184; 57.6%) were identified. CR mechanisms were determined for 146 randomly chosen isolates: the Carba NP test revealed an enzymatic resistance to carbapenems in 109 isolates (out of 146, 74.7%). Produced carbapenemases were evaluated by the NG-Test Carba5, NG-Test OXA-23 immunochromatographic assays and PCR. Carbapenemase-producing (CP) Enterobacterales expressed blaOXA-48-like, blaNDM-like and blaVIM-like genes and CP-Pseudomonas expressed blaIMP-like and blaVIM-like genes, whereas CP-Acinetobacter expressed blaOXA-23-like genes. The NG-Test Carba5 results were confirmed by PCR sequencing and revealed several variants, such as NDM-19, VIM-62 and OXA-162, never described so far in Lebanon. Isolates with discordant results were sequenced by WGS and highlighted novel variants of the natural oxacillinases of Pseudomonas aeruginosa: blaOXA-50-like genes. Their role in carbapenem resistance should be further studied. Overall, our findings highlight an alarming situation and encourage health care centers to establish performant registration systems that could help in limiting resistance spread.

Differential Binding of Carbapenems with the AdeABC Efflux Pump and Modulation of the Expression of AdeB Linked to Novel Mutations within Two-Component System AdeRS in Carbapenem-Resistant Acinetobacter baumannii.

ABSTRACTResistance-nodulation-division-type efflux system AdeABC plays an important role in carbapenem resistance among Acinetobacter baumannii. However, a knowledge gap is observed regarding the role of its regulator AdeRS in carbapenem-resistant A. baumannii (CRAB). This study effectively combines microbiological analysis with an in-silico structural approach to understand the contribution of AdeRS among CRAB (n = 38). Additionally, molecular docking was performed for the first time to study the interaction of FDA-approved carbapenems and pump inhibitor PAβN with the open and closed structure of AdeB at the three binding sites (periplasmic, proximal, distal). It was observed that open conformation of AdeB facilitates the binding of carbapenems and PAβN at entrance and proximal sites compared to the closed conformation. PAβN was found to block carbapenem interacting residues in AdeB, establishing its role as a competitive inhibitor of AdeB substrates. Overexpression of AdeABC was detected by q-RT-PCR among 29% of CRABs, and several mutations within AdeS (GLY186VAL, SER188PHE, GLU121LYS, VAL255ILE) and AdeR (VAL120ILE, ALA136VAL) were detected by sequencing. The sequence and structure-based study of AdeRS was performed to analyze the probable effect of these mutations on regulation of the two-component system (TCS), especially, utilizing its three-dimensional structure. AdeS mutations inhibited the transfer of a phosphate group to AdeR, preventing the binding of AdeR to the intercistronic region, leading to overexpression of AdeABC. The elucidation of the role of mutations in AdeRS improves our understanding of TCS-based regulation. Identification of the key residues of AdeB interacting with carbapenems and PAβN may help in future designing of novel inhibitors.IMPORTANCE AdeABC is an important efflux pump in A. baumannii that plays a role in resistance toward different antibiotics including the “last resort” antibiotic, carbapenem. This pump is regulated by a two-component system, AdeRS. To understand the binding of carbapenems with AdeABC and pump inhibition by PAβN, we analyzed for the first time the possible atomic level interactions of carbapenems and PAβN with AdeB. In the current study, AdeRS-associated novel mutations in clinical A. baumannii are reported for the first time, and a sequence-structure based in-silico approach was used to interpret their role in AdeABC overexpression, leading to carbapenem resistance. None of the previous studies had undertaken both these aspects simultaneously. This study analyzes the open and closed conformation of AdeB, their binding with carbapenems, and key residues involved in it. This helps in visualizing the plausible atomic level causes of pump inhibition driving the discovery of novel inhibitors.

Genetic Factors Associated with Enhanced blaKPC Expression in Tn3/Tn4401 Chimeras.

The expression of the blaKPC gene plays a key role in carbapenem resistance in Enterobacteriaceae However, the genetic regulators of the blaKPC gene have not been completely elucidated, especially the genes in Tn3-Tn4401 chimeras. Two novel Tn3-Tn4401 chimera isoforms were characterized in our hospital, isoform A (CTA), which harbors a 121-bp deletion containing the PX promoter and was present in 22.6% (54/239) of isolates, and isoform C (CTC), which harbors a 624-bp insertion and a P1 promoter deletion and was present in only 1 isolate. The carbapenem MICs of both isoforms were 2-fold or more higher than those of the wild type (Tn3-Tn4401 chimera, CTB), and blaKPC was most highly expressed in CTA. Bioinformatics and 5' rapid amplification of cDNA ends (5' RACE) experiments indicated a novel strong putative promoter, PY, at the 3' end of the ISKpn8 gene. PY mutation nearly abrogated blaKPC expression (P < 0.01) and restored carbapenem susceptibility in all 3 isoforms. Although the mutation of PX or P1 halved blaKPC expression in CTB (P < 0.05), PX deletion caused a 68% increase in blaKPC expression (P = 0.037) in CTA. The level of blaKPC mRNA in CTC was 8-fold higher than that in InCTC, which harbors P1 (P = 0.011). These results suggest that PY is a core promoter of the blaKPC gene in the chimeras and that the deletion of the PX and P1 promoters enhanced gene expression in CTA and CTC, respectively.

Mechanisms of carbapenem resistance in Acinetobacter pittii and Acinetobacter nosocomialis isolates from Thailand.

The emergence of carbapenem resistance in non-baumannii Acinetobacter has increased in clinical settings worldwide. We investigated the prevalence and mechanisms of carbapenem resistance in A. pittii and A. nosocomialis Thai isolates. Acinetobacter calcoaceticus-Acinetobacter baumannii (Acb) complex isolates were identified by gyrB mulitplex PCR. Carbapenem susceptibilities were studied by the agar dilution method and carbapenemase genes were detected by multiplex PCR. Reductions of the outer membrane proteins (OMPs) were evaluated by SDS-PAGE. Overexpressions of efflux pumps were detected by using efflux pump inhibitors and RT-PCR. Of the 346 Acb isolates, 22 and 19 were A. pittii and A. nosocomialis, respectively. The carbapenem resistance rates were 22.7 % in A. pittii and 26.3 % in A. nosocomialis. Three carbapenem-resistant A. pittii carried blaOXA-23. One carbapenem-resistant A. pittii harboured blaOXA-58, while another isolate co-harboured blaOXA-58 and blaIMP-14a. blaOXA-58 was also found in three carbapenem-susceptible A. pittii. Five carbapenem-resistant A. nosocomialis carried blaOXA-23. Eighteen A. pittii isolates carried blaOXA-213-like. Reduced OMPs were found in carbapenem-resistant and -susceptible A. pittii carrying blaOXA-58, but were not detected in carbapenem-resistant A. nosocomialis isolates. Overexpression of adeE was found in carbapenem-resistant A. pittii. No efflux pump genes were present in carbapenem-resistant A. nosocomialis. The major mechanisms of carbapenem resistance in A. pittii and A. nosocomialis were the production of OXA-23 and OXA-58. Overexpression of adeE played a role in carbapenem resistance in A. pittii. Since blaOXA-58 was found in carbapenem-susceptible A. pittii, using carbapenems in the treatment of A. pittii infection should be considered.

Porin Deficiency in Carbapenem-Resistant Enterobacter aerogenes Strains.

The more frequent reports of carbapenem-resistant Enterobacteriaceae have raised the alarm for public health. Apart from the production of carbapenemases, deficiency (decreased or loss of expression) of outer membrane proteins (OMPs) has been proposed as a potentially important mechanism of carbapenem resistance. The aim of the present study was to evaluate the contribution of the major OMPs to carbapenem resistance in Enterobacter aerogenes (CREA) isolates and also investigate the role of small RNAs (sRNAs) in inducing porin-associated permeability defects. The differential expression of OMPs was analyzed in four clinical CREA isolates. omp35 and omp36 genes were further investigated by whole-genome sequencing, induction of meropenem resistance, sRNA overexpression, OMP complementation assays, and reverse transcription-quantitative PCR. All four isolates examined were deficient in omp35 and omp36. Functional restoration of these two genes confirmed their contribution to carbapenem resistance. The meropenem induction assay further revealed that porin deficiency plays a role in carbapenem resistance under antibiotic selection pressure. Single-point mutations in omp36 leading to premature stop codons were detected in two of the isolates. Elevated expression levels of the sRNAs micF and micC were detected in the other two porin-deficient isolates, which were predicted to be potential porin regulators from whole-genome sequencing. Overexpression of micF and micC downregulated the expression of Omp35 and Omp36, respectively. Porin deficiency plays an important role in carbapenem resistance among clinical E. aerogenes isolates under regulation of the sRNAs micC and micF. Furthermore, overexpression of micC and micF had a minor to no impact on carbapenem minimum inhibitory concentrations, and thus, the regulatory mechanism is likely to be complex.

Whole genome and transcriptome analysis reveal MALDI-TOF MS and SDS-PAGE have limited performance for the detection of the key outer membrane protein in carbapenem-resistant Klebsiella pneumoniae isolates.

To detect the outer membrane protein (OMP), which plays a key role in carbapenem resistance, whole-genome and transcriptome analysis of the clinical carbapenem-resistant Klebsiella pneumoniae was carried out. The index strain lacked both OmpK35 and OmpK36, whereas the other strains lacked only OmpK35. After SDS-PAGE, the putative OMP bands were excised and identified as OmpA and OmpK36. MALDI-TOF MS showed peaks at ∼36 and ∼38 kDa that corresponded to OmpA and OmpK36, respectively. In all the strains except YMC2014/03/P345, the ∼38 kDa peaks were present. The K. pneumoniae ATCC 13883 isolate showed three bands on SDS-PAGE and three corresponding peaks on MALDI-TOF MS. The additional third peak at ∼37 kDa corresponding to OmpK35 was observed. To verify OmpK35 peak detection in other K. pneumoniae isolates by MALDI-TOF MS, we analyzed six strains from our laboratory’s strain bank. Whole genome sequence indicated that only two isolates had intact OmpK35. Both MALDI-TOF MS and SDS-PAGE did not show a ∼37 kDa peak or an OmpK35 band as observed in the K. pneumoniae ATCC 13883 isolate. Separation using SDS-PAGE showed a single peak representing OmpA. Therefore, both SDS-PAGE and MALDI-TOF MS were not completely reliable for OMP detection because they fail to detect OmpK35. To the best of our knowledge, this is the first report on the performance of SDS-PAGE and MALDI-TOF MS for the detection of OMP’s using whole-genome and RNA sequencing analyses.

Spatial molecular epidemiology of carbapenem-resistant and New Delhi metallo beta-lactamase (blaNDM)-producing Escherichia coli in the piglets of organized farms in India.

A cross-sectional study was conducted in 10 government-organized pig farms between 2014 and 2016 representing seven states of India to understand the epidemiology of carbapenem resistance in the Escherichia coli. In this study, fecal sample (n = 673) from non-diarrheic (n = 501) and diarrheic (n = 172) piglets were processed for isolation of carbapenem resistant E. coli. Of 673, E. coli isolate (n = 112) was genotyped for confirming the carbapenem resistance and associated virulence factors. Of the 112 isolates, 23 were phenotypically resistant to carbapenem and 8 were carrying the New Delhi metallo beta-lactamase (blaNDM) gene. The carbapenem-resistant isolates also produced extended spectrum beta-lactamases and were multidrug resistant. The PCR-based pathotyping revealed the presence of stx1, stx2, eae and hlyA genes. The enterobacterial repetitive intergenic consensus PCR dendrogram analysis of the isolates yielded three distinct clusters. The statistical analysis revealed no association between carriages of carbapenem-resistant E.coli in different breed of piglets however, location, sex, health status of piglets and age showed significant difference. The spatial analysis with SaTScan helped in identification of carbapenem-resistant clusters. The presence of carbapenem resistant E. coli isolates with virulence genes in the piglet poses a potential public health risk through possible access and spread via the food chain and environment. Efflux pump may also play an important role in carbapenem resistance in piglet E. coli isolates. Furthermore, identification of risk factors in relation to spatial clusters will help in designing preventive strategies for reducing the risk of spread of carbapenem resistant bacteria. 1. Piglets harbor carbapenem resistant E.coli and have great public health significance. 2. Apart from carbapenemase, efflux pump is also important for carbapenem resistance. 3. This is the first report of blaNDM in the piglets from India.

First Detection of GES-5 Carbapenemase-Producing Acinetobacter baumannii Isolate.

This study was conducted to investigate the molecular epidemiology of resistance in Acinetobacter baumannii isolates collected at a hospital in Riyadh, Saudi Arabia, from January through December 2010. Twenty-seven A. baumannii were highly resistant (MIC90 > 256 μg/ml) to ceftazidime, cefepime, and aztreonam. Imipenem resistance was seen in 24 isolates, of which 18 had an minimum inhibitory concentration (MIC) >32 μg/mL. Ciprofloxacin, gentamicin, and amikacin resistance was found in 93%, 52%, and 37% of all the isolates, respectively. Moreover, 8 (30%) isolates showed colistin resistance, and 15 (56%) were found to have MICs ≥4 μg/mL for tigecycline. The frequency of ADC, GES-1, GES-11, and GES-5 were 96.3% (n = 26), 18.5% (n = 5), 11% (n = 3), and 3.7% (n = 1), respectively. OXA-23 was found in 63% (n = 17) of the isolates; ISAba1 was found upstream of OXA-23 in 16. OXA-24/40 was detected in only one strain. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) analysis revealed that the 27 strains were distributed in 8 sequence types (STs) and 16 clonal pulsotypes (A-P). Five singleton STs were identified, including ST15 and ST113-ST116. The emergence of multidrug-resistant A. baumannii is becoming a major concern in Saudi Arabia. Metallo-β-lactamases have no role in carbapenem resistance in this collection. The spread of OXA-23 in our strains occurred across different STs and pulsotypes, unlike what has been observed in many other countries. PFGE typing was more discriminatory than MLST. The high frequency of colistin and tigecycline resistance found in the isolates calls for continuous monitoring. This study describes the first identification of GES-5 conferring carbapenem resistance in A. baumannii.

Rapid detection of Pseudomonas aeruginosa and Acinetobacter baumannii Harboring bla(VIM-2), bla(IMP-1) and bla(OXA-23) genes by using loop-mediated isothermal amplification methods.

BackgroundCarbapenem-resistant Pseudomonas aeruginosa (CRPA) and Acinetobacter baumannii (CRAB) are the leading causes of nosocomial infections. A rapid and sensitive test to detect CRPA and CRAB is required for appropriate antibiotic treatment. We optimized a loop-mediated isothermal amplification (LAMP) assay to detect the presence of blaVIM-2, blaIMP-1, and blaOXA-23, which are critical components for carbapenem resistance.MethodsTwo sets of primers, inner and outer primers, were manually designed as previously described. The LAMP buffer was optimized (at 2mM MgSO4) by testing different concentrations of MgSO4. The optimal reaction temperature and incubation time were determined by using a gradient thermocycler. Then, the optimized blaVIM-2, blaIMP-1, and blaOXA-23 LAMP reactions were evaluated by using 120 P. aeruginosa and 99 A. baumannii clinical isolates.ResultsOnly one strain of the 100 CRPA isolates harbored blaIMP-1, whereas none of them harbored blaVIM-2. These results indicate that the acquisition of blaVIM-2 or blaIMP-1 may not play a major role in carbapenem resistance in Korea. Fifty two strains of the 75 CRAB isolates contained blaOXA-23, but none contained blaVIM-2 and blaIMP-1 alleles.ConclusionsOur results demonstrate the usefulness of LAMP for the diagnosis of CRPA and CRAB.

Allocation of Klebsiella pneumoniae Bloodstream Isolates into Four Distinct Groups by ompK36 Typing in a Taiwanese University Hospital.

The OmpK36 porin plays a role in carbapenem resistance and may contribute to bacterial virulence in Klebsiella pneumoniae. This study aimed to investigate the characteristics of different groups of K. pneumoniae separated by ompK36 typing. Among 226 nonduplicate K. pneumoniae bloodstream isolates collected at a Taiwanese hospital in 2011, four ompK36 types, designated types A, B, C, and D, were identified by PCR in 61, 28, 100, and 36 isolates, respectively; 1 isolate was untypeable. Statistical analysis showed significantly higher rates of antimicrobial resistance (all tested antibiotics except meropenem), extended-spectrum β-lactamases or DHA-1 (47.5% together), Qnr-type quinolone resistance determinants (50.8%), and IncFIIA-type plasmids (49.2%) in group A than in others. Seventeen isolates were identified as belonging to 3 international high-risk clones (4 sequence type 11 [ST11], 10 ST15, and 3 ST147 isolates); all isolates but 1 ST15 isolate were classified in group A. The significant characteristics of group C were hypermucoviscosity (62.0%) and a higher virulence gene content. This group included all serotype K1 (n = 30), K2 (n = 25), and K5 (n = 3) isolates, 6 of 7 K57 isolates, all isolates of major clones associated with pyogenic liver abscesses (29 ST23, 11 ST65, 5 ST86, 7 ST373, and 1 ST375 isolates), and 16 (94.1%) of 17 isolates causing bacteremic liver abscesses. Twelve (42.9%) of the group B isolates were responsible for bacteremic biliary tract infections. Group D was predominant (83.3%) among 12 K20 isolates. This study suggests that most clinical K. pneumoniae isolates can be allocated into four groups with distinct characteristics based on ompK36 types.

ISAba15Inserted into Outer Membrane Protein GenecarOinAcinetobacter baumannii

We identified ISAba15 inserted into the carO gene in an Acinetobacter baumannii isolate. The insert disrupted the lpxD gene, resulting in colistin resistance in A. baumannii. Persistence in carbapenem resistance in A. baumannii isolates with an intact carO gene indicates that loss of the encoded CarO may play a minor role in carbapenem resistance.

Klinik Örneklerden İzole Edilen Acinetobacter baumannii Suşlarında Beta-Laktamaz Kaynaklı Direncin Moleküler Karakterizasyonu

Acinetobacter baumannii is an important cause of nosocomial infections that particularly increase the mortality and the morbidity at the intensive care units of the hospitals. The aims of this study were to evaluate the resistance genes, antibiotic susceptibility and the clonal relations among Acinetobacter strains isolated from clinical samples and to determine the resistance mechanisms related to these bacteria in our hospital. A total of 201 A.baumannii strains isolated from different clinical samples (35.3% from tracheal aspirate, 27.3% from blood, 18.4% from abscess material, 19% from other samples) of 160 inpatients evaluated at the Ibni Sina Hospital Central Bacteriology Laboratory, Ankara University School of Medicine, Turkey from April 2010 to December 2011, were included in the study. Identification of the isolates and their susceptibility testing against amikacin, ciprofloxacin, tetracycline, sulbactam/ampicillin, trimethoprim/sulfametoxazole (SXT), ceftazidime, gentamicin, imipenem, levofloxacin, meropenem, piperacillin/tazobactam, cefoperazone/sulbactam, cefepime and colistin were performed by the automated systems, namely Vitek 2 (bioMérieux, France) and BD Phoenix (Becton Dickinson, USA). The molecular mechanisms of beta-lactamase resistance and the presence of integrons were analyzed by polymerase chain reaction (PCR). Moreover, since blaPER-1 gene is of high frequency in Turkey, it was also investigated in the isolates. Pulsed-field gel electrophoresis (PFGE) was performed to examine the clonal relations between isolates. Our results indicated that multidrug resistance rate of A.baumannii was 94.5% (190/201), while 94% (189/201) of the isolates were susceptible to colistin thus making it the most potent antimicrobial agent, followed by amikacin and SXT with a susceptibility rate of 32%. Twelve colistin-resistant isolates were further investigated with the E-test method (AB Biodisk, Sweden) and found to be colistin-resistant. While the results were negative for the genes responsible from metallo-beta-lactamase production, positive results were obtained for blaOXA genes at various rates (OXA-51 100%; OXA-23 91.5%; OXA-58 7%; OXA-24 2%). PFGE results revealed four different main clones (29 isolates in genotype A, 23 in genotype B, 18 in genotype C and 7 in genotype D) in the study population. No common epidemic isolate was detected. Class 1 integrons which take part in the transfer of resistance genes were detected in 112 (55.7%) isolates. There was no statistically significant difference between the genotype distributions of class 1 integron positive strains (p> 0.05). The relationship between the presence of integron in multidrug resistant isolates and resistance to tetracyclin, SXT, imipenem, meropenem, cefoperazone/sulbactam and cefepime were found to be statistically significant (p< 0.05). Of the isolates 42 (21%) were positive for blaPER- 1 gene and all were resistant to ceftazidime. This study indicated that blaOXA genes found together with blaOXA-51 genes play an important role in carbapenem resistance of A.baumannii strains. Moreover, multidrug resistance is still an important problem in infections caused by A.baumannii and integrons play a role in the transfer of the resistance genes. In conclusion, multidrug resistant A.baumannii strains were common in our hospital and our epidemiologic data would be helpful for further investigations and in therapeutical approaches.

Characterization of a novel Zn²⁺-dependent intrinsic imipenemase from Pseudomonas aeruginosa.

Previous work showed that PA5542 inactivation increases Pseudomonas aeruginosa 59.20 susceptibility to carbapenems. The objective of the current study was to purify PA5542, to determine its role in carbapenem resistance and to analyse the kinetic constants of this putative new β-lactamase. PA5542 was cloned and expressed in Escherichia coli. The enzyme was purified by affinity as a GST fusion protein and, after that, cleaved to remove the GST tag. β-Lactamase activity was measured spectrophotometrically using imipenem as substrate. Susceptibility to antibiotics was determined by Etest. Zn(2+) was added when needed. The expression levels of PA5542, ampC, poxB, mexA and oprD were determined by real-time RT-PCR. Lack of PA5542 increases P. aeruginosa 59.20 susceptibility to carbapenems and its overexpression reduces E. coli susceptibility to these β-lactams. PA5542 is highly conserved in all sequenced P. aeruginosa strains. The clinical isolate 59.20 is resistant to imipenem (MIC >32 mg/L) and to meropenem (MIC 24 mg/L) and presents high-level expression of PA5542 in comparison with the wild-type strain PAO1. Spectrophotometric analyses showed that PA5542 is a Zn(2+)-dependent imipenemase. Analysis of the PA5542 sequence indicates that it does not belong to the classical categories of β-lactamases. PA5542 encodes a new Zn(2+)-dependent imipenemase. The presence of PA5542 in all sequenced P. aeruginosa genomes, maintaining the synteny and without adjacent gene-mobility elements, indicates that it belongs to the P. aeruginosa core genome. High PA5542 expression in 59.20 suggests it may contribute to the resistance to carbapenems of this P. aeruginosa clinical isolate.

Molecular characterization of carbapenem-insensitive Acinetobacter baumannii in Egypt

This study investigated the prevalence of diverse Ambler class β-lactamase-encoding genes in 40 carbapenem-insensitive Acinetobacter baumannii isolates collected from two hospitals in Egypt during the period January-March 2012. The resistance levels to different groups of antimicrobial agents were determined. PCR was used to detect the different Ambler class β-lactamases encoding the following genes: blaTEM, blaSHV, blaCTX-M, blaVEB, blaPER, blaGES, blaVIM, blaIMP, blaSIM, blaSPM, blaGIM, blaNDM, blaADC, blaOXA-23, blaOXA-24, blaOXA-51, and blaOXA-58. ISAba1 and int1 were detected by PCR. The isolates were 100% resistant to amoxicillin-clavulanate, aztreonam, cefepime, cefotaxime, and ceftazidime. Of the isolates, 5% were resistant to colistin, 45% to amikacin, 70% to imipenem, and 85% to ciprofloxacin. The blaADC- and blaOXA-51-like genes were detected in the entire collection. The prevalences of blaOXA-23, blaOXA-24, and blaOXA-58 were 50%, 7.5%, and 5%, respectively. However, the prevalences of blaTEM-, blaPER-, and blaGES-like genes were 87.5%, 55%, and 27.5%, respectively. SHV, CTX-M, VEB, KPC, and MBL encoding genes were not detected. The ISAba1 was found upstream to blaOXA-51, blaOXA-23, and blaADC in 85%, 80%, and 50%, respectively. Of note, 45% (18/40) of the isolates co-produced extended-spectrum β-lactamases (PER and GES) and carbapenemases (OXA-23 and OXA-58). The blaADC-, blaTEM-, blaPER-, blaOXA-23-, and blaGES-like genes were found to be the most prevalent types of β-lactamase-encoding gene in A. baumannii collected from Egypt. A high level of carbapenem resistance is mediated by blaOXA-23, blaOXA-24, and blaOXA-58 (minimum inhibitory concentration (MIC) 32 to >256μg/ml), and a low level of carbapenem resistance is mediated by blaGES (MIC 4-16μg/ml) and by up-regulation of ISAba1-OXA-51 (MIC 1-4μg/ml). Class B MBL was not identified to play a role in carbapenem resistance in A. baumannii isolates from Egypt.

Conformational stability of OXA-51 β-lactamase explains its role in carbapenem resistance of Acinetobacter baumannii

Acinetobacter baumannii, an important nosocomial pathogen, is increasingly becoming resistant to antibiotics including recent β-lactam like imipenem. Production of different types of β-lactamases is one of the major resistance mechanisms which bacteria adapt. We recently reported the presence of a β-lactamase, OXA-51, in clinical strains of A. baumannii in ICUs of our hospital. This study is an attempt to understand the structure–function relationship of purified OXA-51 in carbapenem resistance in A. baumannii. The OXA-51 was cloned, expressed in E. coli Bl-21(DE3) and further purified. The in vitro enzyme activity of purified OXA-51 was confirmed by two independent techniques; in-gel assay and spectrophotometric method using nitrocefin. Further in vivo effect of OXA-51 was followed by transmission electron microscopy of bacterium. Biophysical and biochemical investigations of OXA-51 were done using LC-MS/MS, UV–Vis absorption, fluorescence, circular dichroic spectroscopy and isothermal calorimetry. Native OXA-51 was characterized as 30.6 kDa, pI 8.43 with no disulphide bonds and comprising of 30% α-helix, 27% β-sheet. Secondary structure of OXA-51 was significantly unchanged in broad pH (4–10) and temperature (30–60 °C) range with only local alterations at tertiary structural level. Interestingly, enzymatic activity up to 75% was retained under above conditions. Hydrolysis of imipenem by OXA-51 (km,1 μM) was found to be thermodynamically favourable. In the presence of imipenem, morphology of sensitive strain of A. baumannii was drastically changed, while OXA-51-transformed sensitive strain retained the stable coccobacillus shape, which demonstrates that imipenem is able to kill sensitive strain but is unable to do so in OXA-51-transformed strain. Hence the production of pH- and temperature-stable OXA-51 appears to be a major determinant in the resistance mechanisms adopted by A. baumannii in order to evade even the latest β-lactams, imipenem. It can be concluded from the study that OXA-51 plays a vital role in the survival of the pathogen under stress conditions and thus poses a major threat.

Antibiotic Trapping by Plasmid-Encoded CMY-2 β-Lactamase Combined with Reduced Outer Membrane Permeability as a Mechanism of Carbapenem Resistance in Escherichia coli

A liver transplant patient was admitted with cholangitis, for which meropenem therapy was started. Initial cultures showed a carbapenem-susceptible (CS) Escherichia coli strain, but during admission, a carbapenem-resistant (CR) E. coli strain was isolated. Analysis of the outer membrane protein profiles showed that both CS and CR E. coli lacked the porins OmpF and OmpC. Furthermore, PCR and sequence analysis revealed that both CS and CR E. coli possessed bla(CTX-M-15) and bla(OXA-1). The CR E. coli strain additionally harbored bla(CMY-2) and demonstrated a >15-fold increase in β-lactamase activity against nitrocefin, but no hydrolysis of meropenem was detected. However, nitrocefin hydrolysis appeared strongly inhibited by meropenem. Furthermore, the CMY-2 enzyme demonstrated lower electrophoretic mobility after its incubation either in vitro or in vivo with meropenem, indicative of its covalent modification with meropenem. The presence of the acyl-enzyme complex was confirmed by mass spectrometry. By transformation of the CMY-2-encoding plasmid into various E. coli strains, it was established that both porin deficiency and high-level expression of the enzyme were needed to confer meropenem resistance. In conclusion, carbapenem resistance emerged by a combination of elevated β-lactamase production and lack of porin expression. Due to the reduced outer membrane permeability, only small amounts of meropenem can enter the periplasm, where they are trapped but not degraded by the large amount of the β-lactamase. This study, therefore, provides evidence that the mechanism of "trapping" by CMY-2 β-lactamase plays a role in carbapenem resistance.

Carbapenem-hydrolyzing oxacillinase in high resistant strains of Acinetobacter baumannii isolated from India

Acinetobacter baumannii, a Gram negative bacterium causes nosocomial infections including bacteremia, secondary meningitis and urinary tract infections. Increased resistance of A. baumannii has been global concern. Till recently, carbapenems, latest generation of β-lactams are used for treating infections caused by A. baumannii. Emerging resistance to carbapenem class is an immediate threat to mankind. The objective of present study is to understand the growing carbapenem resistance of A. baumannii. By using iso-electric focusing followed by (in-gel) nitrocefin assay of carbapenem resistant strains of A. baumannii, we could identify three β-lactamases with pIs in the range 5.4–9.5. Expression of the β-lactamase with a pI ≈ 8.5, was found only in very high carbapenem resistant (MIC for imipenem 128 μg/ml) strains. On PCR analysis and sequencing of PCR product, this β-lactamase was confirmed to be OXA-51. Identification of this protein from IEF gel was reconfirmed with the help of Liquid chromatography and Tandem mass spectrometry (LC-MS/MS). Based on the amino acid sequence, OXA-51 found to be a 30 kDa β-lactamase containing conserved functional motifs of class D serine β-lactamase. In the present study, we have established the emergence of OXA-51 in clinical strains of A. baumannii in India which suggests its role in carbapenem resistance.

Plasmid pKpQIL encoding KPC-3 and TEM-1 confers carbapenem resistance in an extremely drug-resistant epidemic Klebsiella pneumoniae strain

An extremely drug-resistant (XDR) clone of KPC-3-producing Klebsiella pneumoniae emerged in Israel in 2006, causing a nationwide outbreak. We aimed to characterize the local KPC-3-encoding plasmid carried by these isolates and study its contribution to antibiotic resistance. Mechanisms of carbapenem resistance were investigated in seven selected isolates (isolated between 2006 and 2008) belonging to the epidemic clone. Isolates underwent MIC testing, and were examined for the presence of KPC, Tn4401, class I integron elements and additional antibiotic resistance genes. Plasmids were analysed by transformation, transconjugation, restriction mapping, curing and complementation experiments. Outer membrane protein (OMP) analysis was performed. OMP analysis did not reveal loss of porins. KPC-3-producing K. pneumoniae isolates possessed various plasmids but all harboured a common self-transmissible 105 kb plasmid, termed pKpQIL, encoding bla(TEM-1) and bla(KPC-3). Curing of pKpQIL led to a complete loss of resistance to cephalosporins and carbapenems, proving its crucial role in carbapenem resistance. Transformation of plasmid pKpQIL into the cured Klebsiella strain resulted in full reconstitution of carbapenem resistance. The presence of all Tn4401 transposon elements located upstream of the KPC-3 gene was detected by PCR and sequencing. pKpQIL lacked additional antibiotic resistance genes. Our findings demonstrate the presence of pKpQIL, a 105 kb KPC-3- and TEM-1-encoding plasmid, in the XDR K. pneumoniae epidemic strain in Israel. pKpQIL is unique and appears consistently in all isolates of this clone over the years. The extensive beta-lactam resistance phenotype of this clone is primarily mediated by this single self-transmissible plasmid.

Antimicrobial Susceptibility and Serotype Distribution of Nontyphoid Salmonella Clinical Isolates in Seven Asian Countries, 2003–2005

the bacteria grown in iron deficit medium. This suggests that the outer membrane protein also plays an important role in carbapenem resistance in Acinetobacter spp. Conclusion: Multiple mechanisms involved for carbapenem resistance in Acinetobacter spp. and therefore, understanding carbapenem resistance mechanisms might be crucial for the development of novel therapeutic strategies. However, it will be an important approach in the near future if one attempt to develop possible targets of new agents to control antimicrobial resistance in nosocomial pathogens such as Acinetobacter spp.