Clinical Enterobacteriaceae Strains Research Articles

Comprehensive evaluation of clinical antimicrobial resistance using impedance-accelerated single-bacterium multiplex screening strategy

Rapid detection of bacterial antibiotic resistance is paramount in clinical settings to enable timely and effective therapeutic interventions. Traditional approaches such as disc diffusion and broth dilution are indeed dependable, however, they suffer from a notable drawback of long assay durations. This is attributed to the inherent delay caused by bacteria's macroscopic growth rate, which often necessitates measurement periods exceeding 20 hours, a timeframe incompatible with the clinical urgency. In this study, we demonstrate a comprehensive strategy on rapid bacterial antimicrobial resistance profiling and testing in clinical samples via a label-free impedance-accelerated single-bacterium screening. This system involves one newly developed parameter that is calculated from the two-dimensional kernel density values of different density regions in bacterial impedance spectra. By evaluating the changes in bacterial populations over a certain period using this new parameter, antimicrobial resistance of individual strains of bacteria can be determined within 20-minute antibiotics exposure. This high-throughput method rapidly and accurately determines the resistance profiles of five clinical Enterobacteriaceae strains against six antibiotic types. During the analysis of the impedance spectrum, we have also observed that antibiotics with different antimicrobial mechanisms have distinct effects on the bacterial impedance profile. This proposed method demonstrates excellent accuracy and rapid response time, providing a novel and practical strategy for next-generation antimicrobial susceptibility testing (AST) and antibiotic management.

Genotypic Distribution and Antimicrobial Susceptibilities of Carbapenemase-Producing Enterobacteriaceae Isolated in Tertiary Care Hospital in South India

Antimicrobial resistance has emerged as a major danger to contemporary medicine around the world. Carbapenems are the highest class of B-lactam drugs which are considered as the most effective and safest antibiotics available. Increasing spread of carbapenemases has been noted across the world which restricts treatment options. The aim of this study was to determine the incidence of carbapenem resistant genotypic pathways in a tertiary care hospital. 130 clinical strains of Enterobacteriaceae were subjected to Kirby-Bauer disk diffusion tests and genotypic methods (PCR) for the identification of the genes NDM, VIM, and OXA-48. Carbapenem resistance was detected in 30% of the isolates by phenotypic methods. These 37 isolates on being subject to PCR showed OXA-48 followed by VIM and NDM as the most frequently isolated genotypes. All isolates had multiple genes encoding carbapenem resistance. Carbapenemases resistance is on the rise and is associated with multi drug resistance pattern. To minimize spread and initiate early appropriate therapy, early detection of carbapenem resistance is essential. Molecular methods remain gold standard for detection.

Efficacy of cephalosporins against enterobacteria isolated from patients with chronic osteomyelitis

Long-term antibiotic therapy, as well as inappropriate use of drugs in the treatment of osteomyelitis, can lead to the appearance of pan-resistant strains. The existing antibiotic prophylaxis regimens for purulent-septic complications are outdated and need to be adjusted. In this regard, it is necessary to monitor the resistance of microorganisms in order to identify ineffective antibacterial drugs. To analyze the resistance profiles of Enterobacteriaceae isolated from patients with chronic osteomyelitis to cephalosporin drugs over a three-year period. The resistance profiles of 912 clinical strains of Enterobacteriaceae were analyzed: Klebsiella pneumoniae (n=349), Proteus sp. (n=208), Escherichia coli (n=176), Enterobacter cloacae (n=179) for the period from 2018-2020 to cephalosporin drugs. In 2018, 66.2% of Enterobacteriaceace were resistant to the 1st generation cephalosporins, in 2019 - 78.7%, in 2020 - 79.5%. Generation II cephalosporins were most active against Proteus sp. bacteria, but a decrease in clinical effect was observed by 2020. Among the third generation cephalosporins in 2018, cefotaxime was most active, but in 2020 the number of resistant strains doubled and amounted to 86.3%. Ceftazidime was active against 47.1% of Enterobacteriaceae isolates in 2018, in 2019 - 45% of strains, in 2020 - 37.2% of bacterial strains. High activity of ceftriaxone was noted only in 2018 against Proteus sp. Preparations of the IV generation in 2018 showed the highest activity against bacteria of the genus Proteus, the least - against bacteria K. pneumoniae. In the period from 2019-2020, a significant decrease in the effectiveness of cefepime was observed.The monitoring of the resistance profiles to antibiotics of the cephalosporin series revealed their low efficacy against Enterobacteriaceae isolated from wounds and fistulas of patients with chronic osteomyelitis, which shows the inexpediency of their empirical use.

Transmission and stable inheritance of carbapenemase gene (blaKPC-2 or blaNDM-1)-encoding and mcr-1-encoding plasmids in clinical Enterobacteriaceae strains

ObjectivesThe aim of this study was to investigate the potential for transmission and heritability of carbapenemase gene (blaKPC-2 or blaNDM-1)-encoding or mcr-1-encoding plasmids in clinical Enterobacteriaceae strains. MethodsPotential for transmission of carbapenemase gene (blaKPC-2 or blaNDM-1)-encoding or mcr-1-encoding plasmids in clinical Enterobacteriaceae strains was tested in three conjugation models, namely filter-mating conjugation in laboratory conditions, a meat product model and the gastrointestinal (GI) tract of rats. Plasmid stability in Enterobacteriaceae strains was also determined. ResultsWe demonstrated that plasmids carrying a carbapenemase gene (blaKPC-2 or blaNDM-1) could be efficiently conjugated to strains carrying the mcr-1 gene and vice versa, and that these plasmids could stably co-exist in clinical Enterobacteriaceae strains. These findings suggest that Enterobacteriaceae can readily acquire phenotypic resistance to both carbapenems and colistin in natural environments such as food products and the GI tract of human and animals. ConclusionGene transfer events are common among members of the Enterobacteriaceae and serve as a key mechanism facilitating adaptation to new environments. Development of innovative strategies and surveillance measures to curtail the dissemination of multidrug resistance plasmids is necessary. Transmission and stable inheritance of these two types of plasmids would lead to the emergence of multidrug-resistant pathogens that are resistant to all currently available last-line antibiotics for Gram-negative bacterial infections.

Molecular detection of plasmid-derived AmpC β-lactamase among clinical strains of Enterobacteriaceae in Bahrain.

BACKGROUND:Enterobacteriaceae with AmpC β-lactamase are multidrug-resistant organisms and represent a significant challenge to patient care. This study aims to determine the prevalence of plasmid-derived AmpC β-lactamase among extended spectrum β-lactamases (ESBL)-producing Enterobacteriaceae strains in Bahrain.METHODS:It was a cross-sectional study. A total of 185 ESBL-producing Enterobacteriaceae isolates were recovered from clinically significant specimens from January 2018 to December 2019. The samples underwent initial screen for cefoxitin resistance by disc diffusion test and subsequent phenotypic confirmation of AmpC production with phenyl boronic acid assays as well as genotypic analysis by multiplex polymerase chain reactions for AmpC subtypes. Drug-resistant features of these clinical isolates were also examined.RESULTS:Twenty-nine ESBL-producing Enterobacteriaceae isolates were cefoxitin resistant. Phenotypic and genotypic analyses confirmed that 8 and 12 cefoxitin-resistant isolates are AmpC positive, respectively. These AmpC producers are multidrug resistant, and Escherichia coli is the dominant strain among them.CONCLUSIONS:Plasmid-mediated spread of AmpC is present in clinically relevant Enterobacteriaceae species in Bahrain. Rational antimicrobial therapy against these multidrug-resistant organisms and continued surveillance of antimicrobial resistance mechanisms among the clinical isolates are recommended for optimal patient care.

Exploring the Potential of CRISPR-Cas9 Under Challenging Conditions: Facing High-Copy Plasmids and Counteracting Beta-Lactam Resistance in Clinical Strains of Enterobacteriaceae.

The antimicrobial resistance (AMR) crisis urgently requires countermeasures for reducing the dissemination of plasmid-borne resistance genes. Of particular concern are opportunistic pathogens of Enterobacteriaceae. One innovative approach is the CRISPR-Cas9 system which has recently been used for plasmid curing in defined strains of Escherichia coli. Here we exploited this system further under challenging conditions: by targeting the blaTEM–1 AMR gene located on a high-copy plasmid (i.e., 100–300 copies/cell) and by directly tackling blaTEM–1-positive clinical isolates. Upon CRISPR-Cas9 insertion into a model strain of E. coli harboring blaTEM–1 on the plasmid pSB1A2, the plasmid number and, accordingly, the blaTEM–1 gene expression decreased but did not become extinct in a subpopulation of CRISPR-Cas9 treated bacteria. Sequence alterations in blaTEM–1 were observed, likely resulting in a dysfunction of the gene product. As a consequence, a full reversal to an antibiotic sensitive phenotype was achieved, despite plasmid maintenance. In a clinical isolate of E. coli, plasmid clearance and simultaneous re-sensitization to five beta-lactams was possible. Reusability of antibiotics could be confirmed by rescuing larvae of Galleria mellonella infected with CRISPR-Cas9-treated E. coli, as opposed to infection with the unmodified clinical isolate. The drug sensitivity levels could also be increased in a clinical isolate of Enterobacter hormaechei and to a lesser extent in Klebsiella variicola, both of which harbored additional resistance genes affecting beta-lactams. The data show that targeting drug resistance genes is encouraging even when facing high-copy plasmids. In clinical isolates, the simultaneous interference with multiple genes mediating overlapping drug resistance might be the clue for successful phenotype reversal.

Searching for the Optimal Treatment for Metallo- and Serine-β-Lactamase Producing Enterobacteriaceae: Aztreonam in Combination with Ceftazidime-avibactam or Meropenem-vaborbactam.

Objective: Metallo-β-lactamase (MBL)-producing Enterobacteriaceae, particularly those that co-harbor serine β-lactamases, are a serious emerging public health threat given their rapid dissemination and the limited number of treatment options. Pre-clinical and anecdotal clinical data support the use of aztreonam in combination with ceftazidime-avibactam against these pathogens, but other aztreonam-based combinations have not been explored. The objective of this study was to evaluate the in vitro activity and compare synergy between aztreonam in combination with ceftazidime-avibactam and meropenem-vaborbactam against serine and MBL-producing Enterobacteriaceae via time-kill analyses. Methods: 8 clinical Enterobacteriaceae strains (4 Escherichia coli and 4 Klebsiella pneumoniae) co-producing NDM and at least one serine β-lactamase were used for all experiments. Drugs were tested alone, in dual β-lactam combinations, and in triple drug combinations against all strains. Results: All strains were resistant to ceftazidime-avibactam and meropenem-vaborbactam and 7/8 (87.5%) strains were resistant to aztreonam. Aztreonam combined with ceftazidime-avibactam was synergistic against all 7 aztreonam-resistant strains. Aztreonam combined with meropenem-vaborbactam was synergistic against all aztreonam-resistant strains with the exception of an OXA-232-producing K. pneumoniae strain. Neither triple combination was synergistic against the aztreonam-susceptible strain. Likewise, neither dual β-lactam combination was synergistic against any strain. Conclusions: These data suggest that aztreonam plus meropenem-vaborbactam has similar activity to aztreonam plus ceftazidime-avibactam against Enterobacteriaceae producing NDM and other non-OXA-48-like serine β-lactamases. Confirmation of these findings in future in vitro and in vivo models is warranted.

AcrAB–TolC Inhibition by Peptide-Conjugated Phosphorodiamidate Morpholino Oligomers Restores Antibiotic Activity in Vitro and in Vivo

Overexpression of bacterial efflux pumps is a driver of increasing antibiotic resistance in Gram-negative pathogens. The AcrAB-TolC efflux pump has been implicated in resistance to a number of important antibiotic classes including fluoroquinolones, macrolides, and β-lactams. Antisense technology, such as peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs), can be utilized to inhibit expression of efflux pumps and restore susceptibility to antibiotics. Targeting of the AcrAB-TolC components with PPMOs revealed a sequence for acrA, which was the most effective at reducing antibiotic efflux. This acrA-PPMO enhances the antimicrobial effects of the levofloxacin and azithromycin in a panel of clinical Enterobacteriaceae strains. Additionally, acrA-PPMO enhanced azithromycin in vivo in a K.pneumoniae septicemia model. PPMOs targeting the homologous resistance-nodulation-division (RND)-efflux system in P.aeruginosa, MexAB-OprM, also enhanced potency to several classes of antibiotics in a panel of strains and in a cell culture infection model. These data suggest that PPMOs can be used as an adjuvant in antibiotic therapy to increase the efficacy or extend the spectrum of useful antibiotics against a variety of Gram-negative infections.

1987. Validation of a MALDI-TOF MS-Based Direct-on-Target Microdroplet Growth Assay (DOT-MGA) for Rapid Detection of Extended-Spectrum β-Lactamase (ESBL) and AmpC in Clinical Enterobacteriaceae Isolates

BackgroundMost phenotypic methods routinely employed for the detection of ESBL and AmpC producing Enterobacteriaceae require 18 hours of incubation. Aiming to offer this clinically relevant information in a shorter time, we developed a MALDI-TOF MS-based direct-on-target microdroplet growth assay (DOT-MGA) as a one-step screening and confirmation panel in accordance with the EUCAST guidelines.MethodsDOT-MGA was performed on 12 clinical Enterobacteriaceae strains displaying resistance against third-generation cephalosporins plus four control strains recommended by EUCAST for detection of ESBL production. Microdroplets (6 µL) containing bacterial suspension and antibiotics (cefpodoxime, cefotaxime, ceftazidime, cefepime with or without clavulanic acid, and/or cloxacillin) in cation-adjusted Mueller–Hinton broth (CA-MHB) were spotted directly onto MBT Biotargets 96 (Bruker Daltonics, Germany). Targets were incubated for 4 hours at 36°C in plastic transport boxes in order to avoid evaporation. Subsequently, culture medium was removed and MALDI-TOF MS of the cells adhered to the target’s surface was performed. The minimum inhibitory concentration (MIC) was considered to be the lowest concentration at which the MALDI Biotyper software (Bruker) provided no species identification. ESBL/AmpC production was defined as an 8-fold or greater decrease of the cephalosporin MIC when combined with clavulanic acid and/or cloxacillin, respectively. A computer-based algorithm was established to interpret the results, which were compared with those of broth microdilution (reference method). Resistance mechanisms of the clinical isolates were assessed by PCR.ResultsThe method correctly identified the ESBL and AmpC production of both control and clinical strains (positive agreement, 100%; negative agreement, 100%), allowing the differentiation of each mechanism even in isolates displaying combined resistance. The results agreed with the characterization of the strains by PCR.ConclusionWe present a rapid method for phenotypic detection of ESBL and AmpC that yields reliable results in a significantly shorter time, thus representing a potentially valuable tool in the routine detection of multidrug-resistant pathogens.Disclosures E. A. Idelevich, Bruker Daltonik: Co-inventor of a pending patent, Licensing agreement or royalty and Speaker honorarium. K. Sparbier, Bruker Daltonik: Employee, Salary. M. Kostrzewa, Bruker Daltonik: Employee, Salary. K. Becker, Bruker Daltonik: Co-inventor of a pending patent, Licensing agreement or royalty and Speaker honorarium.

English

Increasing bacterial resistance to antimicrobial agents has become an issue of concern. A major problem of the treatment of infections caused by Enterobacteriaceae using antibiotics is the emergence of Extended-spectrum β-lactamases (ESBL)-producing Enterobacteriaceae. This study aims to determine the prevalence of ESBL-producing Enterobacteriaceae strains in Ouagadougou, Burkina Faso, and describe their resistance profile to other antibiotics commonly used in the infections treatment. 486 clinical strains of Enterobacteriaceae were obtained from patients attending three health centers in Ouagadougou (Burkina Faso) from November 2014 to October 2015. Biochemical identification was performed and antibiotics susceptibility test was performed using the disk diffusion method. Data was analyzed with the Excel and ANOVA one-way software GraphPad Prism version 5.01 software. Results revealed occurrence of Escherichia coli (60.9%, 194) predominated followed by Klebsiella spp. (22.4%, 109). Antibiotics susceptibility test revealed that 86.8% strains were resistant to amoxicillin, 81.3% to trimethoprim-sulfamethoxazole, 61.9% to ceftriaxone, 58.6% to cefotaxime and 58.4% to cefepime. It was observed that 99.8% were susceptible to imipenem while 16.6% were resistant to fosfomycin and 12.3% to amikacin. However, 38.5% (187/486) of the strains were ESBL-producing, 67.9% (127/187) of which came from Yalgado Ouedraogo University Hospital Center, 23.5% (44/187) from Charles De Gaulle Paediatric University Hospital Center and 8.6% (16/187) from Saint Camille Hospital. This study thus showed a high prevalence of Extended-Spectrum Β-Lactamases producing Enterobacteriaceae strains in Ouagadougou (38.5%). It underlined the need for routine detection and systematic reporting of ESBL strains in different health facilities in Burkina Faso, so that measures could be taken to prevent their spread and treatment failures. Key words: Enterobacteriaceae, Extended-Spectrum Beta-lactamases (ESBL), Burkina Faso.

Characterization of Novel Integrons, In1085 and In1086, and the Surrounding Genes in Plasmids from Enterobacteriaceae, and the Role for attCaadA16 Structural Features during attI1 × attC Integration.

Novel class 1 integrons In1085 and In1086, containing the class D β-lactamase -encoding gene blaOXA, were identified in clinical enterobacterial strains. In this study, we aimed to characterize the genetic contexts of In1085 and In1086, with the goal of identifying putative mechanisms of integron mobilization. Four plasmids, approximately 5.3, 5.3, 5.7, and 6.6 kb, from 71 clinical Enterobacteriaceae strains were found to contain class 1 integrons (In37, In62, In1085, and In1086, respectively). Two of these plasmids, pEco336 and pNsa292, containing In1085 and In1086, respectively, were further characterized by antibiotic susceptibility testing, conjugation experiments, PCR, sequencing, and gene mapping. The OXA-type carbapenemase activities of the parental strains were also assessed. The results revealed that the novel integrons had different genetic environments, and therefore demonstrated diverse biochemical characteristics. Using evolutionary inferences based on the recombination of gene cassettes, we also identified a role for attCaadA16 structural features during attI1 × attC insertion reactions. Our analysis showed that gene cassette insertions in the bottom strand of attCaadA16 in the correct orientation lead to the expression the encoded genes from the Pc promoter. Our study suggests that the genetic features harbored within the integrons are inserted in a discernable pattern, involving the stepwise and parallel evolution of class 1 integron variations under antibiotic selection pressures in a clinical setting.

Study on MICs of Tigecycline in Clinical Isolates of Carbapenem Resistant Enterobacteriaceae (CRE) at a Tertiary Care Centre in North India.

Members of family Enterobacteriaceae are the most common Gram-negative bacteria isolated from clinical samples. Those Enterobacteriaceae which have acquired resistance to all β-lactams antibiotics including the carbapenems are considered as Carbapenem Resistant Enterobacteriaceae (CRE). These CRE isolates are often resistant to most other classes of antimicrobials as well, making their treatment a great challenge. Tigecycline is one of the last resort antimicrobials against such multidrug resistant bacteria. Decreased tigecycline susceptibility mediated by efflux pump systems is being reported in clinical strains of Enterobacteriaceae. Minimum Inhibitory Concentration (MIC) data would prove useful in managing infections by these multidrug resistant bacteria and optimizing use of tigecycline. To evaluate the MIC values of tigecycline against carbapenem resistant Escherichia coli and Klebsiella pneumoniae isolates. This prospective study was carried out from January 2015 to December 2015 at the Department of Microbiology, Era's Lucknow Medical College and Hospital (ELMCH), Lucknow, Uttar Pradesh, India. Antimicrobial susceptibility by disk diffusion (Kirby-bauer) was done for 491 E. coli and K. pneumoniae strains isolated from 1606 samples collected from patients admitted in various wards and ICUs. Imipenem, meropenem and ertapenem 10 μg disks were used for testing of sensitivity to carbapenems. In all isolates, Tigecycline 15 μg (Hi-Media) disk was used to screen for tigecycline resistance. In CRE isolates, MICs of tigecycline were determined by E-test (Ezy MIC TM TG strips, Hi Media) and interpreted using European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2016 guidelines. Out of 491 isolates tested, 186 (37.9%) were found to be CRE showing resistance to at least one of the three carbapenems tested and these included 99 E.coli and 87 K. pneumoniae. Sensitivity pattern of these two bacterial isolates shows a high level of resistance to most classes of antimicrobials. MIC testing for tigecycline was carried out in 144 CRE isolates and tigecycline resistance (MIC >2 μg/ml) was seen in 12 (8.3%) isolates (eight K.pneumoniae and four E. coli). Eight other isolates were found to have MIC of 2 μg/ml and thus the overall prevalence of isolates with decreased susceptibility was 20 (13.9%). A high prevalence of carbapenem resistance coupled with high tigecycline MICs in clinical isolates of E.coli and K. pneumoniae highlights the judicious use of a combination of antimicrobials. Routine in vitro sensitivity testing to evaluate the clinical utility of tigecycline against such resistant Enterobacteriaceae is warranted.

Analyses of Plasmids Harbouring Quinolone Resistance Determinants in Enterobacteriaceae Members.

The aim of this study was to explore the plasmid characteristics of eight clinical Enterobacteriaceae strains containing extended broad spectrum beta-lactamases and plasmid-mediated quinolone resistance. Plasmids were transferred by conjugation or transformation and resistance determinants were investigated by PCR. We showed that at least one plasmid harbouring qnrB or qnrS determinant was transferred by conjugation in five isolates. QepA determinant was confirmed to be on a non-conjugative plasmid. We found at least one beta-lactamase gene in seven of the eight clinical isolates having plasmid-mediated quinolone resistance, which indicated that these two resistance determinants were mostly on the same conjugative plasmids.

Complex class 1 integrons harboring CTX-M-2-encoding genes in clinical Enterobacteriaceae from a hospital in Brazil.

CTX-M enzymes are the most prevalent extended-spectrum beta-lactamases (ESBLs) in Brazil and around the world. The spread of CTX-M lies in their ability to be mobilized by insertion sequences and integrons. This study aimed to identify the mobile genetic structures associated with bla(CTX-M) genes from clinical Enterobacteriaceae strains. Twenty-eight clinical non-clonal Enterobacteriaceae were screened by PCR for the presence of bla(CTX-M) genes and class 1 integrase (int1), and for the association of bla(CTX-M) with class 1 integrons. Plasmid incompatibility groups were assessed by PBRT. Wild-type plasmids were transformed into electrocompetent E. coli, and the S1-PFGE technique was used to verify the presence of high-molecular-weight plasmids in both wild-type strains and E. coli transformants. Sequencing showed that strains carried bla(CTX-M-2) (n = 25) and bla(CTX-M-59) (n = 3) genes inserted into the 3'-end of complex class 1 integrons. Thirteen strains also carried bla(TEM) and bla(SHV) genes. CTX-M-2/59-containing complex class 1 integrons were also present in E. coli transformants. The most frequent Inc groups were IncA/C (n = 10) and IncF (n = 8). Heavy plasmids were observed in both wild-type strains and E. coli transformants. The presence of the same bla(CTX-M-2-group)-containing genetic structure in seven Enterobacteriaceae species isolated at seven hospital wards shows the great mobility potential of complex class 1 integrons. Also, this is the first report of TEM-15, SHV-45, and SHV-55 in Latin America. The genetic environment of bla(CTX-M-2) accounts for their maintenance and spread among Gram-negative bacteria.

Development of a multiplex PCR system and its application in detection of blaSHV, blaTEM, blaCTX-M-1, blaCTX-M-9 and blaOXA-1 group genes in clinical Klebsiella pneumoniae and Escherichia coli strains.

Resistance to β-lactam antibiotics through β-lactamase production by Enterobacteriaceae continues to burden the health-care sector worldwide. Traditional methods for detection of β-lactamases are time-consuming and labor-intensive and newer methods with varying capabilities continue to be developed. The objective of this study was to develop a multiplex PCR (M-PCR) system for the detection of blaSHV, blaTEM, blaCTX-M-1, blaCTX-M-9 and blaOXA-1 group genes and to apply it in clinical Klebsiella pneumoniae and Escherichia coli strains. To do this, we used group-specific PCR primers in singleplex reactions followed by optimization into multiplex reactions. Specificity and sensitivity of the M-PCR were then evaluated using 58 reference strains before its application to detect bla group genes in 203 clinical Enterobacteriaceae strains. PCR amplicons were sequenced to determine the β-lactamase subtypes. The M-PCR system exhibited 100% specificity and sensitivity. In all, 83.7% of K. pneumoniae and 89.8% of E. coli clinical strains harbored bla group genes with 46.9%, 40.1%, 15.0%, 21.1% and 6.1% of K. pneumoniae having blaSHV, blaTEM, blaCTX-M-1, blaCTX-M-9 and blaOXA-1 group genes, respectively, whereas 12.2%, 77.6%, 22.4%, 36.7% and 8.2% of E. coli had blaSHV, blaTEM, blaCTX-M-1, blaCTX-M-9 and blaOXA-1 group genes, respectively. BlaSHV-1, blaSHV-11, blaSHV-27, blaSHV-33, blaSHV-144, blaTEM-1, blaTEM-135, blaOXA-1, blaCTX-M-3, blaCTX-M-9, blaCTX-M-14, blaCTX-M-15, blaCTX-M-27, blaCTX-M-55, blaCTX-M-65 and blaCTX-M-104 were detected. In conclusion, the M-PCR system was efficient and versatile with an advantage of simultaneously detecting all the targeted bla group genes. Hence, it is a potential candidate for developing M-PCR kits for the screening of these genes for clinical or epidemiological purposes.

Modification of the susceptibility of gram-negative rods producing ESβLS to β-lactams by the efflux phenomenon.

The production of β-lactamases is the most important mechanism of Gram-negative rod resistance to β-lactams. Resistance to ceftazidime and cefepime in clinical isolates of Enterobacteriaceae (especially ESβL-positive E. coli and K. pneumoniae) and P. aeruginosa is life-threatening. However, all strains of the above mentioned species possess chromosomally encoded RND efflux pump systems in addition to β-lactamase production. The main goal of this study was to assess the role of efflux pump systems in cefepime and/or ceftazidime resistant phenotypes of ESβL-positive clinical strains of Enterobacteriaceae and P. aeruginosa. The influence of the efflux pump inhibitor PAβN on the minimum inhibitory concentration (MIC) values of tested cephalosporins was species-dependent. Generally, a significant reduction (at least four-fold) of β-lactam MICs was observed in the presence of PAβN only in the case of P. aeruginosa clinical isolates as well as the ESβL-producing transformant PAO1161 ΔampC. The usage of this agent resulted in the restoration of susceptibility to cefepime and/or ceftazidime in the majority of the P. aeruginosa ESβL-positive strains with low and moderate resistance to the above cephalosporins. Moreover, an outer membrane permeabilizing effect in the presence of PAβN was identified. Strain-dependent β-lactamase leakage upon PAβN or β-lactam treatment was demonstrated. The most important observation was the restoration of susceptibility of P. aeruginosa WUM226 to cefepime (MIC decrease from 32 to 4 mg/L) and ceftazidime (MIC decrease from 128 to 4 mg/L) in the presence of PAβN, which occurred despite an almost complete lack of β-lactamase leakage from bacterial cells. In conclusion, these data indicate that RND efflux pumps can modify the susceptibility to β-lactams in Gram-negative rods producing ESβLs. However, this phenomenon occurs only in P. aeruginosa strains and was not observed among E. coli and K. pneumoniae strains, representing the Enterobacteriaceae family.

Evaluation of the βLacta Test, a Rapid Test Detecting Resistance to Third-Generation Cephalosporins in Clinical Strains of Enterobacteriaceae

For decades, third-generation cephalosporins (3GC) have been major drugs used to treat infections due to Enterobacteriaceae; growing resistance to these antibiotics makes the rapid detection of such resistance important. The βLacta test is a chromogenic test developed for detecting 3GC-resistant isolates from cultures on solid media within 15 min. A multicenter prospective study conducted in 5 French and Belgian hospitals evaluated the performance of this test on clinical isolates. Based on antibiotic susceptibility testing, strains resistant or intermediate to cefotaxime or ceftazidime were classified as 3GC resistant, and molecular characterization of this resistance was performed. The rates of 3GC resistance were 13.9% (332/2,387) globally, 9.4% in Escherichia coli (132/1,403), 25.6% in Klebsiella pneumoniae (84/328), 30.3% in species naturally producing inducible AmpC beta-lactamases (109/360), and 5.6% in Klebsiella oxytoca and Citrobacter koseri (7/124). The sensitivities and specificities of the βLacta test were, respectively, 87.7% and 99.6% overall, 96% and 100% for E. coli and K. pneumoniae, and 67.4% and 99.6% for species naturally producing inducible AmpC beta-lactamase. False-negative results were mainly related to 3GC-resistant strains producing AmpC beta-lactamase. Interestingly, the test was positive for all 3GC-resistant extended-spectrum beta-lactamase-producing isolates (n = 241). The positive predictive value was 97% and remained at ≥96% for prevalences of 3GC resistance ranging between 10 and 30%. The negative predictive values were 99% for E. coli and K. pneumoniae and 89% for the species producing inducible AmpC beta-lactamase. In conclusion, the βLacta test was found to be easy to use and efficient for the prediction of resistance to third-generation cephalosporins, particularly in extended-spectrum beta-lactamase-producing strains.

Activity of Tigecycline in Combination with Colistin, Meropenem, Rifampin, or Gentamicin against KPC-Producing Enterobacteriaceae in a Murine Thigh Infection Model

Limited antimicrobials remain active for treating severe infections due to KPC-producing pathogens, and optimal regimens have not been established. In murine thigh infections caused by nine KPC-producing clinical strains of Enterobacteriaceae (meropenem MICs, 1 to 4 μg/ml), we evaluated the activities of tigecycline, colistin, meropenem, rifampin, and gentamicin in single and combination regimens lasting for 24 h and 48 h. Rifampin, tigecycline, and gentamicin were the most effective monotherapies, reducing significantly the CFU counts yielded from thighs infected by 88.9 to 100%, 77.8 to 88.9%, and 66.7 to 88.9% of strains, respectively; meropenem and colistin alone exhibited considerably lower performance (significant CFU reduction in 33.3% and 22.2 to 33.3% of the strains, respectively). The addition of rifampin or gentamicin to tigecycline produced synergistic effect in most strains, while antagonism was observed in 33.3 to 44.4% of the strains when colistin was added to tigecycline and in 44.4 to 55.5% of the strains for meropenem combination with tigecycline. Tigecycline combinations with gentamicin or with rifampin caused higher CFU reductions than did tigecycline plus colistin or plus meropenem with almost all strains. Furthermore, tigecycline plus gentamicin was significantly more effective than tigecycline plus colistin or tigecycline plus meropenem in 33.3 to 44.4% and 55.5 to 66.7% of the strains, respectively, while tigecycline plus rifampin significantly outperformed tigecycline plus colistin and tigecycline plus meropenem in 33.3% and 66.7 to 77.8% of the strains, respectively. Overall, our in vivo study showed that tigecycline plus rifampin or plus gentamicin is a robust regimen against soft tissue infections caused by KPC-producing strains. The combinations of tigecycline with colistin or meropenem should be considered with caution in clinical practice.

Evaluation of the AID ESBL line probe assay for rapid detection of extended-spectrum β-lactamase (ESBL) and KPC carbapenemase genes in Enterobacteriaceae

This study aimed at evaluating the AID ESBL line probe assay for the detection of extended-spectrum β-lactamase (ESBL) and KPC carbapenemase genes in Enterobacteriaceae. The AID ESBL line probe assay was verified for accuracy of its probes using PCR products from clinical ESBL Enterobacteriaceae strains harbouring TEM, SHV and CTX-M ESBL genes and KPC genes and mutant fusion PCR products generated from Enterobacteriaceae strains containing wild-type (wt) TEM and wt SHV. Sensitivity and specificity was determined testing a set of 424 clinical Enterobacteriaceae strains (including 170 strains negative for TEM, SHV, CTX-M and KPC to evaluate the possibility of false positive signals). The line probe assay was shown to detect with 100% accuracy ESBL genes for which oligonucleotide probes are present in the assay. Testing a set of 424 clinical Enterobacteriaceae strains showed 100% sensitivity and specificity for the detection and differentiation of TEM, SHV and CTX-M ESBL genes present in that group. In addition, the line probe assay detected KPC genes accurately. The AID ESBL line probe assay is an accurate and easy-to-use test for the detection of ESBL and KPC genes, which can readily be implemented in the diagnostic laboratory.

Characterising the biology of novel lytic bacteriophages infecting multidrug resistant Klebsiella pneumoniae

BackgroundMembers of the genus Klebsiella are among the leading microbial pathogens associated with nosocomial infection. The increased incidence of antimicrobial resistance in these species has propelled the need for alternate/combination therapeutic regimens to aid clinical treatment. Bacteriophage therapy forms one of these alternate strategies.MethodsElectron microscopy, burst size, host range, sensitivity of phage particles to temperature, chloroform, pH, and restriction digestion of phage DNA were used to characterize Klebsiella phages.Results and conclusionsOf the 32 isolated phages eight belonged to the family Myoviridae, eight to the Siphoviridae whilst the remaining 16 belonged to the Podoviridae. The host range of these phages was characterised against 254 clinical Enterobacteriaceae strains including multidrug resistant Klebsiella isolates producing extended-spectrum beta-lactamases (ESBLs). Based on their lytic potential, six of the phages were further characterised for burst size, physicochemical properties and sensitivity to restriction endonuclease digestion. In addition, five were fully sequenced. Multiple phage-encoded host resistance mechanisms were identified. The Siphoviridae phage genomes (KP16 and KP36) contained low numbers of host restriction sites similar to the strategy found in T7-like phages (KP32). In addition, phage KP36 encoded its own DNA adenine methyltransferase. The φKMV-like KP34 phage was sensitive to all endonucleases used in this study. Dam methylation of KP34 DNA was detected although this was in the absence of an identifiable phage encoded methyltransferase. The Myoviridae phages KP15 and KP27 both carried Dam and Dcm methyltransferase genes and other anti-restriction mechanisms elucidated in previous studies. No other anti-restriction mechanisms were found, e.g. atypical nucleotides (hmC or glucosyl hmC), although Myoviridae phage KP27 encodes an unknown anti-restriction mechanism that needs further investigation.