Clinical Isolates Of Enterobacteriaceae Research Articles

Assessment of Antibiotic Resistance among Clinical Isolates of Enterobacteriaceae in Nepal.

Clinicians face a global challenge treating infections caused by Enterobacteriaceae because of the high rate of antibiotic resistance. This cross-sectional study from the Nepal Armed Police Force Hospital, Kathmandu, Nepal, characterized resistance patterns in Enterobacteriaceae across different antimicrobial classes and assessed incidences of multidrug-resistant (MDR) and extensively drug-resistant (XDR) infections. Enterobacteriaceae from clinical samples were isolated on blood and MacConkey agar, except for urine samples on cysteine lactose electrolyte-deficient agar. To determine antimicrobial susceptibility patterns, including MDR and XDR, the Kirby-Bauer disc diffusion method was used. Statistics were performed using SPSS, v. 17.0. Members of the family were identified in 14.5% (95% CI: 16.2-12.8%) of the total samples (N = 1,617), primarily in urine (54.7%, 128/234), blood (19.7%, 46/234), and sputum (15.0%, 35/234). Escherichia coli (n = 118, 44.2%) was the most predominant bacteria, followed by Citrobacter freundii (n = 81, 30.3%). As much as 95.6% (392/410) of the isolates were penicillin-resistant, whereas only 36.2% (290/801) were carbapenem-resistant. A total of 96 (36.0%) MDR and 98 (36.7%) XDR Enterobacteriaceae were identified. Proteus mirabilis (44.4%, 8/18) predominated MDR cultures, whereas C. freundii (53.1%, 43/81) predominated XDR cultures. Multidrug resistant (38.4%, 71/154) and XDR Enterobacteriaceae (22.7%, 35/154) were chiefly uropathogens. Fluoroquinolone resistance rates in non-MDR, MDR, and XDR isolates were 19.9%, 63.2%, and 96.2%, respectively, whereas cephalosporin resistance rates were 28.6%, 72.9%, and 95.4% and penicillin resistance rates were 67.0%, 97.4%, and 98.0%. One-seventh of patients visiting the hospital were found to be infected with Enterobacteriaceae, and of these patients, at least one-fourth were infected with MDR strains.

Network meta-analysis of antibiotic resistance patterns in gram-negative bacterial infections: a comparative study of carbapenems, fluoroquinolones, and aminoglycosides

IntroductionAntimicrobial resistance poses a grave global threat, particularly with the emergence of multidrug-resistant gram-negative bacterial infections, which severely limit treatment options. The increasing global threat of antimicrobial resistance demands rigorous investigation, particularly concerning multidrug-resistant gram-negative bacterial infections that present limited therapeutic options. This study employed a network meta-analysis, a powerful tool for comparative effectiveness assessment of diverse antibiotics. The primary aim of this study was to comprehensively evaluate and compare resistance patterns among widely used antibiotic classes, namely carbapenems, fluoroquinolones, and aminoglycosides, for combating gram-negative pathogens.MethodsWe searched PubMed, Web of Sciences, Scopus, Scholarly, Medline, Embase, and Cochrane databases up to August 27, 2023. Studies showing antibiotic resistance in clinical isolates of Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii exposed to carbapenems, fluoroquinolones, and aminoglycosides were included. This study determined treatment-specific resistance percentages and ranked these treatments based on resistance using a random-effects network meta-analysis technique. To investigate the impact of the study and pathogen features, subgroup and meta-regression analyses were performed. Risk ratios and 95% confidence intervals (CIs) were calculated using a network meta-analysis (NMA) incorporating both direct and indirect evidence. Clinical improvement, cure, microbiological eradication, and death from any cause were the primary outcomes. Nephrotoxicity was a secondary result.ResultsThe analysis included 202 publications and 365,782 gram-negative isolates. The NMA included data from 20 studies and 4,835 patients. Carbapenems had the lowest resistance rates throughout the pathogen spectrum, with resistance percentages of 17.1, 22.4, and 33.5% for Enterobacteriaceae, P. aeruginosa, and A. baumannii, respectively. For the same infections, aminoglycosides showed resistance rates of 28.2, 39.1, and 50.2%, respectively. Fluoroquinolones had the highest resistance rates at 43.1, 57.3, and 65.7%, respectively. Unexpectedly, resistance to all three antibiotic classes has increased over time, with multidrug resistance being the most prevalent.ConclusionThis extensive network meta-analysis provides an overview of the patterns of resistance throughout the world and how they are changing. The most effective choice is still carbapenems, but the increasing resistance highlights the critical need for multimodal therapies to protect antibiotic effectiveness against these powerful gram-negative infections.

Resistance of Clinical Isolates of Escherichia coli and Klebsiella pneumoniae in “Boucle du Mouhoun, Burkina Faso”: one year's Experience in Antibiotic Resistance Surveillance

Introduction: Escherichia coli and Klebsiella pneumoniae account for a large proportion of clinically isolated pathogenic bacteria. However, their resistance to antibiotics is increasingly becoming a global health threat. The aim of this study was to describe the current antibiotic resistance profile of these two species. Materials and method: This was a retrospective descriptive study at the Dedougou regional hospital. The results of antibiotics susceptibility testing of non-redundant clinical isolates of Enterobacteriaceae were used. Bacteria were isolated and identified using standard bacteriology methods. The antibiogram was performed by the Kirby-Bauer method and the interpretation was made according to the recommendations of the Antibiogram Committee of the French Microbiology Society (CASFM 2017). Data were entered into WHOnet 2018 and analysed using EPI-INFO 7.2.4.0. Results: A total of 138 non-redundant Enterobacteriaceae strains were isolated, of which almost 90% were E. coli (75.4%) and Klebsiella pneumoniae (13.8%). The most frequent resistance was observed with amoxicillin + clavulanic acid (81.3%), ceftriaxone (66.7%) and cotrimoxazole (82.9%). E. coli showed very high resistance to ampicillin (95.2%). Relatively moderate to high resistance was also observed with ciprofloxacin 69.1% and gentamicin 39%. The most active antibiotics were imipenem and cefoxitin, with resistance frequencies of 2.4% and 5.7% of all strains respectively. ESBL-producing strains were the most frequently encountered phenotypes (59.3%), followed by high-level penicillinases (19.5%). 3GC resistance was associated to ESBL production in almost 90% of cases, and both ciprofloxacin and gentamicin resistance were significantly associated with 3GC resistance (p<0.001). Conclusion: Escherichia coli and Klebsiella pneumoniae are the main enterobacteria isolated in clinics. The production of extended-spectrum beta-lactamases is their main mechanism of resistance to beta-lactam antibiotics.

Cefotaxime/sulbactam plus gentamicin as a potential carbapenem- and amikacin-sparing first-line combination for neonatal sepsis in high ESBL prevalence settings.

Infection with ESBL-producing Enterobacteriaceae infection is ubiquitous in some neonatal ICUs and increasing levels of antibiotic resistance are a cause for urgent concern. Delineation of bacterial and viral sepsis can be challenging, often leading to patients receiving empirical antibiotics without or whilst waiting for a definitive causal diagnosis. Empirical therapy is often dependent on broad-spectrum 'Watch' antibiotics, contributing to further resistance. ESBL-producing Enterobacteriaceae clinical isolates found to have caused neonatal sepsis and meningitis underwent a detailed in vitro screening including susceptibility testing, chequerboard combination analysis and hollow-fibre infection model dynamic analyses using combinations of cefotaxime, ampicillin and gentamicin in combination with β-lactamase inhibitors. Additivity or synergy was found for all antibiotic combinations against seven Escherichia coli and three Klebsiella pneumoniae clinical isolates. Cefotaxime or ampicillin plus sulbactam combined with gentamicin was able to consistently inhibit the growth of ESBL-producing isolates at typical neonatal doses, and the combination cleared the hollow-fibre infection model system of organisms resistant to each agent alone. The combination of cefotaxime/sulbactam and gentamicin was consistently bactericidal at clinically achievable concentrations (Cmax of 180, 60 and 20 mg/L for cefotaxime, sulbactam and gentamicin, respectively). The addition of sulbactam to cefotaxime or ampicillin to the typical first-line empirical therapy could obviate the need for carbapenems and amikacin in settings with high ESBL-infection prevalence.

Aktifitas Antibiotik Ceftazidime-Avibactam Dan Ceftaroline Secara In Vitro Terhadap Isolat Klinis Di Pekanbaru

Ceftaroline and Ceftazidime–avibactam are relatively new antibiotics that show good effectiveness against antibiotic-resistant pathogens. Both of these antibiotics are now available worldwide, but reports of resistance to them are also starting to emerge. This study aimed to evaluate the in vitro activity of the antibiotic Ceftazidime-avibactam combination of 100 clinical isolates consisting of Enterobacteriaceae and Pseudomonas aeruginosa bacteria and to evaluate the antibiotic effect of Ceftaroline against 100 clinical isolates of Enterobacteriaceae and Staphylococcus sp. This study is observational with cross-sectional study design. The study was conducted at the microbiology laboratory of a private hospital at Pekanbaru Riau in Januari-June 2021. There were 100 clinical isolates tested with Ceftaroline and Ceftazidime-avibactam. The sensitivity to Ceftaroline and Ceftazidime-avibactam were reported as a number isolate and percentage. The results showed the sensitivity of bacteria K. pneumoniae, E. coli, Enterobacter sp, and P. aeruginosa, to Ceftazidime-Avibactam were 87%, 90%, 66%, and 82%, respectively. The sensitivity of the antibiotic Ceftaroline to the bacteria K. pneumoniae, E. coli, Enterobacter sp and Staphylococcus sp. were 39%, 54%, 36.4%, 67% and 75%, respectively. Based on these results, the antibiotic ceftazidime avibactam can be used for the treatment of suitable pathogenic infections because of its fairly good sensitivity (>80%) to Enterobacteriaceae and P. aeruginosa bacteria. Ceftaroline antibiotics are not recommended to be used as a therapeutic option, because of the high resistance to Enterobacteriaceae and Staphylococcus sp. Further research is needed to determine the molecular mechanism of resistance to Ceftaroline antibiotics

Prevalence of carbapenemase-producing Enterobacteriaceae from human clinical samples in Ethiopia: a systematic review and meta-analysis

IntroductionCarbapenemase-producing Enterobacteriaceae are by far the most public health and urgent clinical problems with antibiotic resistance. They cause longer hospital stays, more expensive medical care, and greater mortality rates. This systematic review and meta-analysis aimed to indicate the prevalence of carbapenemase-producing Enterobacteriaceae in Ethiopia.MethodsThis systematic review and meta-analysis was conducted based on Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines. Electronic databases like PubMed, Google Scholar, CINAHL, Wiley Online Library, African Journal Online, Science Direct, Embase, ResearchGate, Scopus, and the Web of Sciences were used to find relevant articles. In addition, the Joanna Briggs Institute quality appraisal tool was used to assess the quality of the included studies. Stata 14.0 was used for statistical analysis. Heterogeneity was assessed by using Cochran’s Q test and I2 statistics. In addition, publication bias was assessed using a funnel plot and Egger’s test. A random effect model was used to estimate the pooled prevalence. Sub-group and sensitivity analysis were also done.ResultsThe overall pooled prevalence of carbapenemase-producing Enterobacteriaceae in Ethiopia was 5.44% (95% CI 3.97, 6.92). The prevalence was highest [6.45% (95% CI 3.88, 9.02)] in Central Ethiopia, and lowest [(1.65% (95% CI 0.66, 2.65)] in the Southern Nations and Nationalities People Region. In terms of publication year, 2017–2018 had the highest pooled prevalence [17.44 (95% CI 8.56, 26.32)] and 2015–2016 had the lowest [2.24% (95% CI 0.87, 3.60)].ConclusionThis systematic review and meta-analysis showed a high prevalence of carbapenemase-producing Enterobacteriaceae. So, to alter the routine use of antibiotics, regular drug susceptibility testing, strengthening the infection prevention approach, and additional national surveillance on the profile of carbapenem resistance and their determining genes among Enterobacteriaceae clinical isolates are required.Systematic review registrationPROSPERO (2022: CRD42022340181).

AcrAB-TolC Efflux Pump Mediated Resistance to Carbapenems among Clinical Isolates of Enterobacteriaceae

AcrAB-TolC is a resistance nodulation division type of efflux pump present in Enterobacteriaceae. It non-specifically effluxes antibiotics out of the bacterial cell, thus conferring drug resistance. Increase in the expression of the AcrAB-TolC efflux pump increases resistance to antibiotics. We aimed to study the expression levels of acrA and acrB that encodes AcrAB-TolC efflux pump, to understand efflux pump mediated resistance to carbapenem among clinical isolates of Enterobacteriaceae obtained from various clinical samples. Additionally, co -production of carbapenemase was also detected in the isolates demonstrating efflux pump mediated resistance to carbapenems. A total of 127 carbapenem resistant clinical isolates of Enterobacteriaceae, isolated from a tertiary care hospital were included in the study. An efflux pump inhibition (EPI) assay with reserpine, an efflux pump inhibitor, was performed to screen for isolates exhibiting efflux pump activity. Real Time Reverse Transcriptase qPCR was performed to detect the mRNA over expression levels of acrA and acrB that encodes AcrAB-TolC efflux pump. The control strains K. pneumoniae BAA2146 and E. coli AcrB were used. EPI assay with carbapenem showed that 56 /127(44%) isolates were screen positive indicating efflux pump activity. A total of 12 isolates showed 101 to 107 increase in the expression of both acrA and acrB when compared with the controls indicating a strong efflux pump activity, in addition to producing carbapenemase. The study highlights the role of efflux pump AcrAB-TolC in conferring resistance to carbapenem among clinical isolates of Enterobacteriaceae.

Automated antimicrobial susceptibility testing and antimicrobial resistance genotyping using Illumina and Oxford Nanopore Technologies sequencing data among Enterobacteriaceae.

Whole-genome sequencing (WGS) enables the molecular characterization of bacterial pathogens. We compared the accuracy of the Illumina and Oxford Nanopore Technologies (ONT) sequencing platforms for the determination of AMR classes and antimicrobial susceptibility testing (AST) among 181 clinical Enterobacteriaceae isolates. Sequencing reads for each isolate were uploaded to AREScloud (Ares Genetics) to determine the presence of AMR markers and the predicted WGS-AST profile. The profiles of both sequencing platforms were compared to broth microdilution (BMD) AST. Isolates were delineated by resistance to third-generation cephalosporins and carbapenems as well as the presence of AMR markers to determine clinically relevant AMR classes. The overall categorical agreement (CA) was 90% (Illumina) and 88% (ONT) across all antimicrobials, 96% for the prediction of resistance to third-generation cephalosporins for both platforms, and 94% (Illumina) and 91% (ONT) for the prediction of resistance to carbapenems. Carbapenem resistance was overestimated on ONT with a major error of 16%. Sensitivity for the detection of carbapenemases, extended-spectrum β-lactamases, and plasmid-mediated ampC genes was 98, 95, and 70% by ONT compared to the Illumina dataset as the reference. Our results highlight the potential of the ONT platform’s use in clinical microbiology laboratories. When combined with robust bioinformatics methods, WGS-AST predictions may be a future approach to guide effective antimicrobial decision-making.

Mating pair stabilization mediates bacterial conjugation species specificity

Bacterial conjugation mediates contact-dependent transfer of DNA from donor to recipient bacteria, thus facilitating the spread of virulence and resistance plasmids. Here we describe how variants of the plasmid-encoded donor outer membrane (OM) protein TraN cooperate with distinct OM receptors in recipients to mediate mating pair stabilization and efficient DNA transfer. We show that TraN from the plasmid pKpQIL (Klebsiella pneumoniae) interacts with OmpK36, plasmids from R100-1 (Shigella flexneri) and pSLT (Salmonella Typhimurium) interact with OmpW, and the prototypical F plasmid (Escherichia coli) interacts with OmpA. Cryo-EM analysis revealed that TraNpKpQIL interacts with OmpK36 through the insertion of a β-hairpin in the tip of TraN into a monomer of the OmpK36 porin trimer. Combining bioinformatic analysis with AlphaFold structural predictions, we identified a fourth TraN structural variant that mediates mating pair stabilization by binding OmpF. Accordingly, we devised a classification scheme for TraN homologues on the basis of structural similarity and their associated receptors: TraNα (OmpW), TraNβ (OmpK36), TraNγ (OmpA), TraNδ (OmpF). These TraN-OM receptor pairings have real-world implications as they reflect the distribution of resistance plasmids within clinical Enterobacteriaceae isolates, demonstrating the importance of mating pair stabilization in mediating conjugation species specificity. These findings will allow us to predict the distribution of emerging resistance plasmids in high-risk bacterial pathogens.

The Dynamics of Antimicrobial Resistance among Enterobacteriaceae Isolates in Russia: Results of the 2012-2018 INFORM and ATLAS International Program Studies.

Background: The increasing prevalence of multidrug-resistant Enterobacteriaceae limits the range of active antimicrobial agents, thus worsening clinical outcomes. The objective of this study was to identify the trends in antimicrobial resistance for Enterobacteriaceae in Russia using the databases for the International Network for Optimal Resistance Monitoring (INFORM) and Antimicrobial Testing Leadership and Surveillance (ATLAS) studies between 2012 and 2018. Methods: This subanalysis was performed for 3811 non-duplicate clinical isolates of Enterobacteriaceae to evaluate the in vitro activity of the main classes of antibiotics against relevant clinical isolates from hospitalized patients with complicated infections of different anatomical locations. Results: The lowest susceptibility was observed for colistin (0%), ampicillin (16.4%), and ampicillin/sulbactam (31.1%), whereas the best susceptibility was observed for all combinations containing avibactam (>96%). Among individual antimicrobials, doripenem (3.2%), tigecycline (1.6%), and meropenem (5.9%) exhibited the lowest resistance. Important trends included the decreasing resistance of Enterobacteriaceae to glycylcyclines and the increasing resistance to aminoglycosides and carbapenems. K. pneumoniae strains were most aggressive in terms of the percentage of strains having multidrug resistance (8.3–18.3%, depending on location) and the percentage of ESBL-positive strains (44.8–86.8%). Conclusions: The current patterns and trends of antimicrobial resistance in different bacterial species should be taken into consideration for timely updating of clinical guidelines and local treatment protocols to ensure effective antimicrobial therapy.

The Emergence of Carbapenem Resistant Enterobacteriaceae Producing GIM-1 and SIM-1 Clinical Isolates in Khartoum-Sudan.

PurposeThe aim of this study was to detect multidrug resistant GIM-1 and SIM-1 producing Enterobacteriaceae clinical isolates from hospitalized patients across three Khartoum State Teaching Hospitals, Sudan.Patients and MethodsFrom May 2018 to October 2019, Enterobacteriaceae clinical isolates from inpatients admitted to different Khartoum state hospitals. Genes for carbapenemase (GIM-1 and SIM-1) were amplified by polymerase chain reaction (PCR). Agar dilution method was used to determine MICs for imipenem and meropenem after antimicrobial susceptibility testing.ResultsFive (1.29%) isolates of Enterobacteriaceae [2 (0.51%) Escherichia coli isolates produce GIM-1, 2 (0.51%) Klebsiella pneumoniae isolates (one [0.25%] of each produce of GIM-1 and of SIM-1), and 1 (0.25%) Enterobacter cloacae isolate produce GIM-1]. Susceptibility profiling of the isolates showed a low-level resistance to imipenem and meropenem MICs (8, 16 and 32 μg/mL). It also had resistance to ampicillin, extended-spectrum cephalosporin’s, aztreonam, and amoxicillin-clavulanate and with the two K. pneumoniae strains showing resistance to colistin.ConclusionWe report the emergence of four GIM-1 producing Enterobacteriaceae strains and one strain of SIM-1 producing K. pneumoniae genes, isolated from hospitalized patients, with a high resistance pattern to antimicrobial agents. Whole-genome sequencing (WGS) is necessary for precise identification of clonal diversity backgrounds of acquired carbapenemase genes in diagnostic laboratories as the number of cases of carbapenem resistant Enterobacteriaceae infection increases annually.

Phenotypic Detection of ESBL, AmpC, MBL, and Their Co-occurrence among MDR Enterobacteriaceae Isolates.

Background Emergence of extended-spectrum beta-lactamases (ESBLs), AmpC β-lactamases, and metallo-β lactamases (MBL), and their co-existence among members of Enterobacteriaceae pose newer diagnostic and therapeutic challenges. The present study examines the ESBL, AmpC, and MBL production by various phenotypic methods and their co-occurrence among the multidrug-resistant (MDR) Enterobacteriaceae clinical isolates. Materials and Methods Four hundred non-repetitive Enterobacteriaceae clinical isolates were collected from the Central Referral Hospital, Sikkim. The isolates were used for identification and their antibiotic susceptibility tests were performed according to the Clinical and Laboratory Standard Institute (CLSI) guidelines. ESBL was detected by double-disc synergy test (DDST) and phenotypic confirmatory disc-diffusion test (PCDDT), AmpC detection by AmpC E-test, and boronic acid disc diffusion (BD) test. MBL was detected using the imipenem–imipenem/EDTA disc and carba-NP tests. Results Around 76% were considered MDR. ESBL was seen in 58% and 50.4% based on DDST and phenotypic confirmation disc-diffusion test (PCDDT), respectively. AmpC was detected in 11.8% and 13.1% using a commercial E-test and boronic acid test, respectively. MBL were identified in 12.8% and 14.8% based on MBL imipenem-EDTA and carba-NP tests, respectively. Co-occurrence of ESBL and AmpC, ESBL and MBL, AmpC and MBL was seen in 5.2%, 11.5%, 1.3%, respectively, whereas a combination of these three β-lactamases was observed in only 0.3% of 304 MDR isolates. Conclusion The findings highlight a high prevalence of β-lactamases and their co-production among the Enterobacteriaceae , mainly in Klebsiella pneumoniae and Escherichia coli isolates. The study further highlights the necessity to identify the MDR β-lactamases stains for effective therapy in severe as well as mild bacterial infections, thereby enabling to reduce the risk of MDR in hospital and community settings.

Systematic Review of Plasmid AmpC Type Resistances in Escherichia coli and Klebsiella pneumoniae and Preliminary Proposal of a Simplified Screening Method for ampC.

Beta-lactamase (BL) production is a major public health problem. Although not the most frequent AmpC type, AmpC-BL is increasingly isolated, especially plasmid AmpC-BL (pAmpC-BL). The objective of this study was to review information published to date on pAmpC-BL in Escherichia coli and Klebsiella pneumoniae, and on the epidemiology and detection methods used by clinical microbiology laboratories, by performing a systematic review using the MEDLINE PubMed database. The predictive capacity of a screening method to detect AmpC-BL using disks with cloxacillin (CLX) was also evaluated by studying 102 Enterobacteriaceae clinical isolates grown in CHROMID ESBL medium with the addition of cefepime (FEP), cefoxitin (FOX), ertapenem (ETP), CLX, and oxacillin with CLX. The review, which included 149 publications, suggests that certain risk factors (prolonged hospitalization and previous use of cephalosporins) are associated with infections by pAmpC-BL-producing microorganisms. The worldwide prevalence has increased over the past 10 years, with a positivity rate ranging between 0.1 and 40%, although AmpC was only detected when sought in a targeted manner. CMY-2 type has been the most prevalent pAmpC-BL-producing microorganism. The most frequently used phenotypic method has been the double-disk synergy test (using CLX disks or phenyl-boronic acid and cefotaxime [CTX] and ceftazidime) and the disk method combined with these inhibitors. In regard to screening methods, a 1-µg oxacillin disk with CLX showed 88.9% sensitivity, 100% specificity, 100% positive predictive value (PPV), 98.9% negative predictive value (NPV), and 98.9% validity index (VI). This predictive capacity is reduced with the addition of extended-spectrum beta-lactamases, showing 62.5% sensitivity, 100% specificity, 100% PPV, 93.5% NPV, and 94.1% VI. In conclusion, there has been a worldwide increase in the number of isolates with pAmpC-BL, especially in Asia, with CMY-2 being the most frequently detected pAmpC-BL-producing type of microorganism. Reduction in its spread requires routine screening with a combination of phenotypic methods (with AmpC inhibitors) and genotypic methods (multiplex PCR). In conclusion, the proposed screening technique is an easy-to-apply and inexpensive test for the detection of AmpC-producing isolates in the routine screening of multidrug-resistant microorganisms.

Prevalence and Molecular Characterization of Extended Spectrum β-Lactamase and Carbapenemase-Producing Enterobacteriaceae Isolates from Bloodstream Infection Suspected Patients in Addis Ababa, Ethiopia.

BackgroundProduction of Extended spectrum beta-lactamase (ESBL) and Carbapenemase is the most common strategy for drug resistance in clinical isolates of Enterobacteriaceae. This study was conducted to determine the magnitude of ESBL and Carbapenemase production (CPE) among clinical isolates of Enterobacteriaceae causing bloodstream infections (BSI) in Ethiopia.MethodsA cross-sectional study was performed from September 2018 to January 2019 in Ethiopia. A total of 2397 BSI suspected patients were enrolled and blood culture was performed using a BacT/Alert instrument in combination with conventional methods for identification. After antimicrobial susceptibility test, phenotypic confirmation of ESBLs was done by combined disc-diffusion. Meanwhile carbapenemase production was done by modified carbapenem inactivation method. Multiplex PCR was conducted to detect the presence of blaCTX-M,blaSHV, blaTEM, blaKPC and blaNDM genes.ResultsA total of 104 (4.3%) Enterobacteriaceae were isolated from 2397 BSI suspected patients. Klebsiella pneumoniae (55/104, 52%) was the predominant isolate followed by E. coli, (19.2%, 20/104) and K.oxytoca (17.3%, 18/104). ESBL and carbapenemase production were observed from 70 (67.3%, 57.4 −76.2% at 95% CI) and 8 (7.7%, 3.4–14.6% at 95% CI) isolates respectively. The highest frequency of ESBL and carbapenemase production was observed in K. pneumoniae 78.2% (43/55) and 9.1% (5/55), respectively. All the 70 isolates confirmed as ESBL producers harbored at least one of the ESBL genes and the majority of them carried multiple beta-lactamase genes (84.3%), where blaCTX-M, type was the most predominant (67.3%). Similarly, the entire eight isolates positive for carbapenemase carried blaNDM but none of them carried blaKPC.ConclusionIn our study, the rate of ESBL production among BSI-causing Enterobacteriaceae was alarming and most of the isolates carried multiple types of ESBL genes. A significant magnitude of CPE isolates causing BSI was recorded.

Multiplex PCR: A Molecular Modality for Rapid Detection of CTX-M-15, AAD A1 and QNR S1 Genes among Drug Resistant Clinical Isolates of Enterobacteriaceae in Kanchipuram, South India

Multidrug resistance is a potential threat to the mankind. The prevalence rate of multidrug resistant bacteria is worrisome and has gained significant attention globally. Hence it becomes imperative for clinical laboratories to identify these multidrug resistant pathogens for appropriate therapy. It is equally important to detect the resistance mechanisms which would aid in understanding and development of new therapeutic options. This study was carried out to identify the prevalence of multidrug resistant Enterobacteriaceae from clinical samples recovered from Kanchipuram district. The study was also implicated in designing of novel primers using primer3 tool and standardizing a multiplex PCR targeting ctxm- 15, aadA1, qnrS1 genes in the isolates. Of the 60 clinical isolates studied, 14, 13 and 12 isolates harbored ctx-m-15, qnrS1 gene and aadA1genes respectively. Among these, 2 isolates carried both ctx-m-15 and qnrS1 genes and 3 isolates were found to carry both ctx-m-15 and aadA1 genes. 1 isolate was found to be positive for both aadA1 and qnrS1 genes. None of these isolates harbored all the three targeted genes qnrS1 and aadA1, ctx-m-15. The present study will aid in detecting drug resistant genes like ctx-m-15, aadA1, qnrS1 in clinical isolates with higher sensitivity and specificity and will also reduce the cost of reaction than that required for uniplex PCR. The prevalence rate of drug resistant genes among Kanchipuram district alerts us for the measures needed to be undertaken to control the spread of drug resistant genes in community.

Intracellular Transposition and Capture of Mobile Genetic Elements following Intercellular Conjugation of Multidrug Resistance Conjugative Plasmids from Clinical Enterobacteriaceae Isolates.

ABSTRACTMobile genetic elements (MGEs) are often associated with antimicrobial resistance genes (ARGs). They are responsible for intracellular transposition between different replicons and intercellular conjugation and are therefore important agents of ARG dissemination. Detection and characterization of functional MGEs, especially in clinical isolates, would increase our understanding of the underlying pathways of transposition and recombination and allow us to determine interventional strategies to interrupt this process. Entrapment vectors can be used to capture active MGEs, as they contain a positive selection genetic system conferring a selectable phenotype upon the insertion of an MGE within certain regions of that system. Previously, we developed the pBACpAK entrapment vector that results in a tetracycline-resistant phenotype when MGEs translocate and disrupt the cI repressor gene. We have previously used pBACpAK to capture MGEs in clinical Escherichia coli isolates following transformation with pBACpAK. In this study, we aimed to extend the utilization of pBACpAK to other bacterial taxa. We utilized an MGE-free recipient E. coli strain containing pBACpAK to capture MGEs on conjugative, ARG-containing plasmids following conjugation from clinical Enterobacteriaceae donors. Following the conjugative transfer of multiple conjugative plasmids and screening for tetracycline resistance in these transconjugants, we captured several insertion sequence (IS) elements and novel transposons (Tn7350 and Tn7351) and detected the de novo formation of novel putative composite transposons where the pBACpAK-located tet(A) is flanked by ISKpn25 from the transferred conjugative plasmid, as well as the ISKpn14-mediated integration of an entire 119-kb, blaNDM-1-containing conjugative plasmid from Klebsiella pneumoniae.IMPORTANCE By analyzing transposition activity within our MGE-free recipient, we can gain insights into the interaction and evolution of multidrug resistance-conferring MGEs following conjugation, including the movement of multiple ISs, the formation of composite transposons, and cointegration and/or recombination between different replicons in the same cell. This combination of recipient and entrapment vector will allow fine-scale experimental studies of factors affecting intracellular transposition and MGE formation in and from ARG-encoding MGEs from multiple species of clinically relevant Enterobacteriaceae.

Detection of OqxAB Efflux Pumps, a Multidrug-Resistant Agent in Bacterial Infection in Patients Referring to Teaching Hospitals in Ahvaz, Southwest of Iran.

Antibiotic resistance mechanisms in Enterobacteriaceae are causative agents of global health problems. Bacterial infections due to multidrug resistance (MDR) may be mediated by the overexpression of efflux pumps. In this study, we investigated the prevalence of oqxA and oqxB genes as two encoding agents of efflux pumps and the determination of antibiotic resistance rate in clinical isolates of Enterobacteriaceae. In this study, 100 Enterobacteriaceae isolates collected from different clinical specimens of infectious patients, such as wounds, urine, blood, discharge, and abscesses except stool, were examined. Identification of the isolates was performed using standard biochemical tests such as TSI, citrate, urea, lysine, SIM, MR-VP, and gas production. The antimicrobial susceptibility test was carried out by the Kirby–Bauer disk diffusion method according to CLSI guidelines, and finally, the oqxA and oqxB genes were detected by the PCR method. Among 100 Enterobacteriaceae isolates, Escherichia coli and Enterobacter gergoviae were the most common isolates with 71% and 20%, respectively. Also, the lowest isolates belonged to Enterobacter cloacae (3%) and Klebsiella pneumoniae (1%). Out of 100 Enterobacteriaceae isolates, 37 isolates (37%) were positive for at least one of oqxA or oqxB genes, while both of these genes were detected among 12% of them. oqxAB genes were detected in 8 cases of 20 (40%) Enterobacter gergoviae and 4 cases of 71 (5.7%) E. coli isolates. The antimicrobial susceptibility test showed that all isolates (100%) were susceptible to imipenem, while the maximum resistance to piperacillin, ceftriaxone, and cefotaxime were 69%, 55%, and 55%, respectively. Also, the results of this study showed that antibiotic resistance in Enterobacteriaceae isolates caused by oqxAB genes is increasing among patients in Iran. Therefore, identification of resistant isolates and antibiotic monitoring programs are essential to prevent the spread of MDR isolates.

Molecular Surveillance and Assessment of Ceftolozane/Tazobactam Resistance with Common β-Lactam Antibiotics and β-Lactamase Genes

Ceftolozane/tazobactam (c/t) is a potent β-lactam antibiotic which combines the fifth generation cephalosporin ceftolozane and tazobactam, a β-lactamase inhibitor. The c/t combination therapy was approved in 2014 for the treatment of multidrug resistant (MDR) Enterobacteriaceae, especially intra-abdominal and urinary tract infections. The aim of this study is to assess c/t activity and to examine the association of c/t resistance with four common β-lactamase resistance genes found in clinical Enterobacteriaceae isolates collected from mainly urinary tract infections in an agricultural region in California (USA) between 2013-2020. We tested 993 Extended Spectrum β-lactamases (ESBL) producing Enterobacteriaceae isolates (885 E. coli, 94 K. pneumoniae, 14 other) for c/t susceptibility by Kirby-Bauer disk diffusion and screened using PCR for four common resistance genes with β-lactamase activity(blaTEM, blaOXA, blaSHV, and blaCTX-M) for 855 of the isolates. We also investigated co-resistance of c/t and nine other β-lactam antibiotics. We found that most isolates were susceptible to c/t (58.3%), while 38.5% showed intermediate resistance, and 3.2% were resistant. We also found that K. pneumoniae isolates were more resistant to c/t than E. coli isolates, and that c/t may be a good alternative to carbapenems, in that that some carbapenem resistant isolates were susceptible to c/t. Genotypic analysis showed blaSHV and blaCTX-M are independently associated with elevated c/t resistance. Although c/t demonstrated strong activity against Enterobacteriaceae, the high percentage of isolates with intermediate susceptibility emphasizes the need for close monitoring and continued surveillance for c/t resistance among ESBLs

Optimization of a lysis method to isolate periplasmic proteins from Gram-negative bacteria for clinical mass spectrometry.

Clinical mass spectrometry requires a simple step process for sample preparation. This study aims to optimize the method for isolating periplasmic protein from Gram-negative bacteria and apply to clinical mass spectrometry. The Klebsiella pneumoniae carbapenemase (KPC)-producing E. coli standard cells were used for optimizing the osmotic shock (OS) lysis method. The supernatant from OS lysis was analysed by LC-MS/MS and MALDI-TOF MS. The effectiveness of the OS lysis method for KPC-2-producing Enterobacteriaceae clinical isolates were then confirmed by MALDI-TOF MS. The optimized OS lysis using KPC-2 producing E. coli standard cells showed a high yield of KPC-2 protein and enriches periplasmic proteins. Compared with other lysis methods, the detection sensitivity of KPC-2 protein significantly increased in MALDI-TOF MS analysis. Nineteen clinical isolates were validated by MALDI-TOF MS using the OS method, which also showed higher detection sensitivity compared to other lysis method (e.g., 1.5% n-octyl-β-D-glucopyranoside) (p<0.001). This study provides a straightforward, rapid, affordable, and detergent-free method for the analysis of periplasmic proteins from Enterobacteriaceae clinical isolates. This approach may contribute to MS-based clinical diagnostics.

Effect of multiple, compatible plasmids on the fitness of the bacterial host by inducing transcriptional changes.

Bacteria that acquire plasmids incur a biological cost. Despite this fact, clinical Enterobacteriaceae isolates commonly contain multiple co-existing plasmids harbouring carbapenemase genes. Six different plasmids carrying blaNDM-1, blaNDM-5, blaCTX-M-15, blaKPC-2, blaOXA-181 and blaOXA-232 genes were obtained from Klebsiella pneumoniae and Escherichia coli clinical isolates. Using the E. coli DH5α strain as recipient, 14 transconjugants with diverse plasmid combinations (single or double plasmids) were generated. For each of these, the effects of plasmid carriage on the bacterial host were investigated using in vitro and in vivo competition assays; additionally, the effects were investigated in the context of biofilm formation, serum resistance and survival inside macrophages. Transcriptomic changes in single- and double-plasmid recipients were also investigated. Increased in vitro and in vivo competitiveness was observed when two plasmids carrying blaNDM-1 and blaOXA-232 were co-introduced into the host bacteria. However, DH5α::pNDM5 + pOXA232 and other double-plasmid recipients did not show such competitiveness. DH5α::pNDM5 + pOXA181 did not show any fitness cost compared with a plasmid-free host and single-plasmid transconjugants, while both the double-plasmid recipients with pCTXM15 or pKPC2 exhibited a fitness burden. The double-plasmid recipient DH5α::pNDM1 + pOXA232 also exhibited increased biofilm formation, serum resistance and survival inside macrophages. Transcriptomic analysis revealed that the genes of DH5α::pNDM1 + pOXA232 involved in metabolic pathways, transport and stress response were up-regulated, while those involved in translation were down-regulated. Our study suggests that bacterial strains can gain fitness through the acquisition of multiple plasmids harbouring antibiotic resistance genes, which may be mediated by transcriptomic changes in the chromosomal genes of the bacterial host.