Coli Clinical Isolates Research Articles

Evaluation of the Activities of Commonly Dispensed Antibiotics at Bayero University Kano Health Care Services Unit

There is a growing concern about the effectiveness of antimicrobials in developing countries. Multifaceted factors contribute to the escalation of antibiotic resistance. The counterfeiting of antibiotic drugs is one facet of this. Antibiotics are the most counterfeited medicines worldwide. However, the WHO Committee for the review of Medicines accentuates surveillance checks even after the marketing of drugs or at the dispensing point. This study evaluated the activities of commonly dispensed antibiotics at Bayero University Kano health care service pharmacy unit. Fourteen brands of different antibiotics with the active ingredients amoxicillin, azithromycin, cefixime, ceftriaxone, ofloxacin, ciprofloxacin, and doxycycline were used. The antibiotic susceptibility testing was carried out using the Kirby Bauer disc diffusion method on clinical isolates of Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae. Typed strains of E. coli (ATCC 25923) and S. aureus (ATCC 25922) were also tested. Ciprofloxacin and ofloxacin-containing formulations had efficacy against all the clinical isolates and typed organisms. All organisms except K. pneumoniae were sensitive to azithromycin. Ceftriaxone was active only against E. coli (both clinical isolate and typed organism). About 71% of the antibiotics tested were not sensitive to the tested organisms. The inactivity observed may be due to the counterfeit or substandard nature of the antibiotics, ineffective storage, or the development of resistance. This necessitates more research to determine the extent of the problem and its causes at a large scale.

Evolution of ceftazidime-avibactam and cefiderocol resistance in ST131-H30R1-Escherichia coli isolates with KPC-3 mutants and application of FTIR biotyping.

Ceftazidime-avibactam and cefiderocol represent two of the few alternatives for infections by KPC-producing Enterobacterales. We reported the emergence of resistance to both ceftazidime-avibactam and cefiderocol in a KPC-producing ST131-Escherichia coli (KPC-ST131-Ec) clinical isolate. Antimicrobial susceptibility testing, Fourier-transform infrared (FTIR) spectroscopy, whole-genome sequencing, and cloning experiments were performed. A KPC-49-Ec isolate resistant to ceftazidime-avibactam (MICCZA > 16/4 mg/L) and susceptible to cefiderocol (MICFDC: 2 mg/L) was recovered in a blood sample from an oncologic patient hospitalized in the medical ICU (June 2019) during ceftazidime-avibactam treatment. After 44 days, a KPC-31-Ec resistant to both ceftazidime-avibactam and cefiderocol (MICCZA > 16/4 mg/L, MICFDC: 8 mg/L) was found in a rectal sample during a second cycle of ceftazidime-avibactam treatment. Both KPC-49 (R163S) and KPC-31 (D179Y) were detected in the epidemic ST131-H30R1-Ec high-risk clone and showed a phenotype resembling that of ESBL producers. FTIR spectroscopy managed to differentiate cefiderocol-susceptible and resistant ST131-Ec isolates, and these from others belonging to different clones. After cloning and transformation experiments, KPC-49 and KPC-31 were responsible for ceftazidime-avibactam resistance (MICCZA > 16/4 mg/L) and decreased carbapenem MICs (MICMER ≤ 0.12 mg/L, MICIMI ≤ 1 mg/L). KPC-31 was also shown to be associated with increased MICs of cefiderocol (twofold and threefold dilutions over KPC-3 and KPC-49, respectively). However, mutations in proteins participating in outer membrane stability and integrity, such as TolR, could have a more relevant role in cefiderocol resistance. The effects of ceftazidime-avibactam and cefiderocol co-resistance in clinical isolates of Enterobacterales producing KPC mutants make their identification challenging for clinical laboratories.IMPORTANCEThroughout four admissions in our hospital of a single patient, different KPC-3 variants (KPC-3, KPC-49, and KPC-31) were found in surveillance and clinical ST131-Escherichia coli isolates, after prolonged therapies with meropenem and ceftazidime-avibactam. Different patterns of resistance to cefiderocol and ceftazidime-avibactam emerged, accompanied by restored carbapenem susceptibility. The inability to detect these variants with some phenotypic methods, especially KPC-31 by immunochromatography, and the expression of a phenotype similar to that of ESBL producers, posed challenge to identify these variants in the clinical microbiology laboratory. Molecular methods and whole-genome sequencing are necessary and new techniques able to cluster or differentiate related isolates could also be helpful; this is the case of Fourier-transform infrared spectroscopy, which managed in our study to discriminate isolates by cefiderocol susceptibility within ST131, and those from the non-ST131 ones.

Bothrops snake venom L-amino acid oxidases impair biofilm formation of clinically relevant bacteria

The present work addressed the abilities of two L-amino acid oxidases isolated from Bothrops moojeni (BmooLAAO-I) and Bothrops jararacussu (BjussuLAAO-II) snake venoms to control the growth and prevent the biofilm formation of clinically relevant bacterial pathogens. Upon S. aureus (ATCC BAA44) and S. aureus (clinical isolates), BmooLAAO-I (MIC = 0.12 and 0.24 μg/mL, respectively) and BjussuLAAO-II (MIC = 0.15 μg/mL) showed a potent bacteriostatic effect. Against E. coli (ATCC BAA198) and E. coli (clinical isolates), BmooLAAO-I (MIC = 15.6 and 62.5 μg/mL, respectively) and BjussuLAAO-II (MIC = 4.88 and 9.76 μg/mL, respectively) presented a lower extent effect. Also, BmooLAAO-I (MICB50 = 0.195 μg/mL) and BjussuLAAO-II (MICB50 = 0.39 μg/mL) inhibited the biofilm formation of S. aureus (clinical isolates) in 88% and 89%, respectively, and in 89% and 53% of E. coli (clinical isolates). Moreover, scanning electron microscopy confirmed that the toxins affected bacterial morphology by increasing the roughness of the cell surface and inhibited the biofilm formation. Furthermore, analysis of the tridimensional structures of the toxins showed that the surface-charge distribution presents a remarkable positive region close to the glycosylation motif, which is more pronounced in BmooLAAO-I than BjussuLAAO-II. This region may assist the interaction with bacterial and biofilm surfaces. Collectively, our findings propose that venom-derived antibiofilm agents are promising biotechnological tools which could provide novel strategies for biofilm-associated infections.

Review on β-lactams resistance among Klebsiella pneumoniae and Escherichia Coli clinical isolates

Review on β-lactams resistance among Klebsiella pneumoniae and Escherichia Coli clinical isolates

Impact of short chain fatty acids (SCFAs) on antimicrobial activity of new β-lactam/β-lactamase inhibitor combinations and on virulence of Escherichia coli isolates

In a healthy gut microbiota, short chain fatty acids (SCFAs) are produced. The antibacterial action of SCFAs against intestinal pathogens makes them useful for ensuring the safety of food and human health. In this study, we aimed to assess the in vitro inhibitory activity of SCFAs, and to report, for the first time, their impact on the activity of new β-lactam/β-lactamase inhibitor combinations. The minimum inhibitory concentrations of acetic, propionic, and butyric acids were determined against E. coli clinical isolates recovered from gastrointestinal infections. Cefoperazone/sulbactam, ceftazidime/avibactam and cefepime/enmetazobactam are new β-lactam/β-lactamase inhibitor combinations that were studied for their combined therapeutic effects. Also, the effects of pH and concentration of SCFAs were evaluated on in vitro bacterial growth and expression of genes encoding for motility, adhesion, invasion, and biofilm formation. SCFAs were tested at concentrations of 12 mM at pH 7.4 (ileum-conditions), in addition to 60 mM and 123 mM, at pH 6.5 (colon-conditions). The tested SCFAs showed the same MIC (3750 μg ml−1 ≃ 60 mM) against all isolates. Furthermore, the addition of SCFAs to the tested β-lactam/β-lactamase inhibitor combinations greatly restored the susceptibility of the isolates. SCFAs had significant effect on bacterial growth and virulence in a pH and concentration-dependent manner; low ileal concentration potentiated E. coli growth, while higher colonic concentration significantly suppressed growth and down-regulated the expression of virulence genes (fliC, ipaH, FimH, BssS). Therefore, the significant inhibitory effect of colonic SCFAs on β-lactam/β-lactamase inhibitor combinations might lead to the development of promising treatment strategies.

668. Presence of the Narrow-Spectrum OXA-1 Beta-Lactamase Enzyme is not Associated with Elevated Ceftolozane-Tazobactam MIC Values among ESBL-Producing Escherichia coli Clinical Isolates (CANWARD, 2011-2018)

Abstract Background Beta-lactam-beta-lactamase inhibitor combinations have been proposed as an alternative to carbapenems for the treatment of infections caused by extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli as an antimicrobial stewardship initiative. However, in a recent randomized trial evaluating patients with bacteremia, presence of the narrow spectrum OXA-1 beta-lactamase enzyme among ESBL-producing Enterobacterales was associated with higher piperacillin-tazobactam MICs, and in turn excess mortality among patients treated with piperacillin-tazobactam relative to meropenem. The purpose of this study was to determine whether the in vitro activity of ceftolozane-tazobactam versus ESBL-producing E. coli is similarly compromised by the presence of OXA-1. Methods E. coli clinical isolates were obtained from patients evaluated at hospitals across Canada (January 2011 to December 2018) as part of an ongoing national surveillance study (CANWARD). ESBL production was confirmed using the Clinical and Laboratory Standards Institute phenotypic method. Susceptibility testing was carried out using custom broth microdilution panels, and all isolates underwent whole genome sequencing for beta-lactamase gene detection. Results In total, 485 ESBL-producing E. coli identified as part of the CANWARD study were included. The majority of isolates (91.3%; 443/485) harbored a CTX-M ESBL enzyme. OXA-1 was present in 39.6% (192/485) of isolates. OXA-1 was detected in 62.5% (187/299) of isolates with a CTX-M-15 ESBL enzyme versus only 2.7% (5/186) of isolates with other ESBL enzyme types. Ceftolozane-tazobactam MIC50 and MIC90 values were identical (0.25 µg/mL and 1 µg/mL, respectively) for the isolate subsets with and without OXA-1. Overall, 97.4% and 96.9% of isolates with and without OXA-1 remained susceptible (CLSI breakpoint) to ceftolozane-tazobactam. Conclusion The presence of OXA-1 among ESBL-producing E. coli clinical isolates was not associated with an elevation in MIC values for ceftolozane-tazobactam. These data support further evaluation of ceftolozane-tazobactam as an alternative to carbapenems for the treatment of infections caused by ESBL-producing E. coli, as is being done with the MERINO-3 trial. Disclosures George Zhanel, PhD, Merck: Grant/Research Support.

1675. Activity of Gepotidacin Against Escherichia coli Isolates from Community-acquired Urinary Tract Infections Collected Between 2019-2021 in the United States

Abstract Background Gepotidacin is a novel, bactericidal, first-in-class triazaacenaphthylene antibiotic that inhibits bacterial DNA replication by a distinct mechanism of action that confers activity against most strains of target pathogens, such as Escherichia coli, Staphylococcus saprophyticus, and Neisseria gonorrhoeae, including those resistant to current antibiotics. This study reports on the in vitro activity of gepotidacin and other oral antibiotics tested against E. coli clinical isolates collected from patients with UTIs for a gepotidacin UTI global surveillance study as part of the SENTRY Antimicrobial Surveillance Program. Methods A total of 1,978 E. coli isolates were collected between 2019-2021 from 47 medical centers within the US. All isolates were cultured from urine specimens collected from patients seen in emergency and outpatient medical services representative of community-acquired infections. Bacterial identifications were confirmed by MALDI-TOF. Isolates were tested for susceptibility by CLSI methods at a central laboratory (JMI Laboratories). MIC results for oral antibiotics licensed for the treatment of uUTI, multidrug-resistant (MDR), and extended-spectrum β-lactamase (ESBL) subsets were interpreted per CLSI criteria. Results Gepotidacin (MIC50/90, 2/4 mg/L) displayed good activity against 1,978 E. coli isolates, with 98.3% of all observed gepotidacin MICs ≤4 mg/L (Table). Susceptibility (S) rates for other oral agents tested against these isolates were: amoxicillin-clavulanate (83.7%S), ampicillin (50.9%S), ciprofloxacin (79.1%S), fosfomycin (99.7%S), mecillinam (94.2%S), nitrofurantoin (98.2%S), and trimethoprim-sulfamethoxazole (71.3%S). When tested against the drug-resistant subsets, gepotidacin maintained similar MIC50/90 values (1-2/4 mg/L). Gepotidacin was also active against ESBL and MDR E. coli isolates, inhibiting 94.7% and 95.9%, respectively, at gepotidacin concentrations ≤ 4 mg/L. Conclusion Gepotidacin demonstrated potent in vitro activity against contemporary community-acquired E. coli urine isolates. This activity was maintained among isolates demonstrating resistance to other oral standard of care antibiotics including ESBL, FQ-R, and MDR E. coli. Disclosures SJ Ryan Arends, PhD, AbbVie: Grant/Research Support|GSK: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support|Shionogi: Grant/Research Support Nicole E. Scangarella-Oman, MS, GlaxoSmithKline plc.: Employee and shareholder Lindsey Tholen, BS (ASCP), GSK: Board Member|GSK: work for hire|Shionogi: Grant/Research Support Jennifer M. Streit, BS, MT(ASCP), Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Shionogi: Grant/Research Support Rodrigo E. Mendes, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support|Office for Assistant Secretary of Defense for Health Affairs: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support|Spero Therapeutics: Grant/Research Support.

Molecular Identification of CTX-M and SHV Betalactamase Genes in Escherichia Coli Clinical Isolates in Isfahan

Molecular Identification of CTX-M and SHV Betalactamase Genes in Escherichia Coli Clinical Isolates in Isfahan

Pharmacodynamic evaluation of piperacillin/tazobactam against extended-spectrum β-lactamase (ESBL)-producing versus non-ESBL-producing Escherichia coli in a hollow-fibre infection model

Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli are a global public-health concern. We evaluated the pharmacodynamic activity of piperacillin/tazobactam (TZP) dosing regimens against ESBL-producing versus non-ESBL-producing E. coli. Five E. coli clinical isolates were obtained from Bangladesh. Broth microdilution and whole-genome sequencing (WGS) were performed on the five studied isolates. Three TZP-susceptible ESBL-producing and two non-ESBL-producing E. coli were exposed to TZP regimens of 4.5 g every 6 h (q6h) and every 8 h (q8h) as a 30-min infusion in a dynamic hollow-fibre infection model over 7 days. The extent of bacterial killing was ∼4-5 log10 CFU/mL against ESBL-producing and non-ESBL-producing E. coli with TZP q6h and q8h regimens over the first 8 h. Bacterial killing was similar between two of three ESBL-producing (CTAP#168 and CTAP#169) and two non-ESBL-producing (CTAP#179 and CTAP#180) E. coli clinical isolates over the course of the experiment. ESBL-producing CTAP#173 E. coli was poorly killed (∼1 log) compared with two non-ESBL-producing E. coli over 168 h. WGS revealed that ESBL-producing E. coli isolates co-harboured multiple antimicrobial resistance genes such as blaCTX-M-15, blaEC, blaOXA-1, blaTEM-1 and aac(6')-Ib-cr5. Overall, TZP q6h and q8h dosing regimens attained >3 log bacterial kill against all ESBL-producing or non-ESBL-producing E. coli within 24 h and maintained and prevented the emergence of resistance through the end of the experiment. In conclusion, TZP standard regimens resulted in similar bacterial killing and prevented the emergence of resistance against CTX-M-15-type ESBL-producing and non-ESBL-producing E. coli clinical isolates.

Molecular and Phenotypic Characterization of a Multidrug-Resistant Escherichia coli Coproducing OXA-232 and MCR-1.1 in Argentina

The spread of carbapenem-resistant Enterobacterales has raised concern in clinical settings due to the limited therapeutic options available. OXA-48-like enzymes are still sporadic in South America. The aim of this study was to characterize a multidrug-resistant Escherichia coli isolate from a hospitalized patient in Buenos Aires city. The isolate was characterized phenotypically by determination of its susceptibility pattern, synergistic and colorimetric tests, and molecularly, by PCR, whole genome sequencing, and plasmid analysis. It belonged to ST-744, phylogroup A, and serotype O162/O89: H9. It remained susceptible to ceftazidime, meropenem, aminoglycosides, trimethoprim/sulfamethoxazole, and tigecycline. The presence of blaOXA-232 harbored by a nonconjugative plasmid ColKp3, and blaCTX-M-14, mcr-1.1, and fosL1 in 2 conjugative plasmids, together with their genetic environment, was revealed. To the best of our knowledge, this is the first report of the coproduction of the enzyme OXA-232 and the mcr-1.1 gene in an E. coli clinical isolate in South America in a patient who had not received colistin therapy.

Determination of Pathophysiological Anti-Diarrhoeal Activity of the Aqueous Stem Bark Extract of Ficus platyphylla

This study was motivated by the report of a survey of herbal remedies used by Fulani herdsmen in the management of cow diarrhoea in Plateau State, Nigeria. The report identified F. platyphylla stem bark amongst major herbs. Activity of aqueous stem extract of F. platyphylla against diarrhoea caused by physiological changes and by pathological infections was herein determined. Thirty albino rats were fasted for 18 hours. They were grouped into six and induced with diarrhoea using castor oil. The first two groups received distilled water (1 ml) and loperamide (5 mg/kg), while other groups were administered plant extract 50, 100, 200 and 400 mg/kg body weight respectively. The severity of diarrhoea was assessed hourly for 4 hrs. Gastrointestinal motility experiment was carried out using atropine sulphate (5 mg/kg). In vitro anti-diarrhoeal test was carried out against E. coli clinical and laboratory isolates versus antibiotics. The extract inhibited defecation moderately, and regulated frequency of wet stool better than loperamide, the lowest dose (50 mg/kg b. w.) competed favourably with the loperamide. This dose produced the most remarkable activity considering the latent period, total stool frequency, purging indices and percentage inhibition of defecation. The dose showed remarkable activity in the propulsion of charcoal meal through gastrointestinal tract as displayed in the inhibition of diarrhoea. The results of in vitro anti-diarrhoeal test against E. coli showed that the plant extract was inactive on the microorganism. The results from the present study validated the anti-diarrhoeal potential of F. platyphylla via its physiological action.

Proteomic Analysis of the Colistin-resistant E. coli Clinical Isolate: Explorations of the Resistome.

Antimicrobial resistance is a worldwide problem after the emergence of colistin resistance since it was the last option left to treat carbapenemase-resistant bacterial infections. The mcr gene and its variants are one of the causes for colistin resistance. Besides mcr genes, some other intrinsic genes are also involved in colistin resistance but still need to be explored. The aim of this study was to investigate differential proteins expression of colistin-resistant E. coli clinical isolate and to understand their interactive partners as future drug targets. In this study, we have employed the whole proteome analysis through LC-MS/MS. The advance proteomics tools were used to find differentially expressed proteins in the colistin-resistant Escherichia coli clinical isolate compared to susceptible isolate. Gene ontology and STRING were used for functional annotation and protein-protein interaction networks, respectively. LC-MS/MS analysis showed overexpression of 47 proteins and underexpression of 74 proteins in colistin-resistant E. coli. These proteins belong to DNA replication, transcription and translational process; defense and stress related proteins; proteins of phosphoenol pyruvate phosphotransferase system (PTS) and sugar metabolism. Functional annotation and protein-protein interaction showed translational and cellular metabolic process, sugar metabolism and metabolite interconversion. We conclude that these protein targets and their pathways might be used to develop novel therapeutics against colistin-resistant infections. These proteins could unveil the mechanism of colistin resistance.

1216. Presence of the Narrow-Spectrum OXA-1 Beta-lactamase Enzyme Is Associated with Elevated Piperacillin-Tazobactam MIC Values Among ESBL-producing Escherichia coli Clinical Isolates (CANWARD, 2007-2018)

BackgroundThe clinical outcome of patients with bacteremia due to an extended-spectrum beta-lactamase (ESBL)-producing member of the family Enterobacteriaceae who are treated with piperacillin-tazobactam appears to depend, at least in part, on the piperacillin-tazobactam MIC. The purpose of this study was to determine whether there is any association between the MIC of piperacillin-tazobactam and presence of the narrow spectrum OXA-1 beta-lactamase enzyme among ESBL-producing Escherichia coli.Methods E. coli clinical isolates were obtained from patients evaluated at hospitals across Canada (January 2007 to December 2018) as part of an ongoing national surveillance study (CANWARD). ESBL production was confirmed using the Clinical and Laboratory Standards Institute phenotypic method. Susceptibility testing was carried out using custom broth microdilution panels, and all isolates underwent whole genome sequencing for beta-lactamase gene detection.ResultsIn total, 671 ESBL-producing E. coli were identified as part of the CANWARD study. The majority of isolates (92.0%; 617/671) harbored a CTX-M ESBL enzyme. CTX-M-15 (62.3%; 418/671), CTX-M-27 (13.9%; 93/671), and CTX-M-14 (13.4%; 90/671) were the most common variants identified. The narrow spectrum OXA-1 beta-lactamase enzyme was present in 42.6% (286/671) of isolates. OXA-1 was detected in 66.3% (277/418) of isolates with a CTX-M-15 ESBL enzyme versus only 3.6% (9/253) of isolates with other ESBL enzyme types. The piperacillin-tazobactam MIC50 and MIC90 values were 8 µg/mL and 32 µg/mL for isolates that possessed the OXA-1 enzyme versus 2 μg/mL and 8 µg/mL for those that did not. The percentage of ESBL-producing E. coli isolates that were inhibited by a piperacillin-tazobactam MIC of ≤8 μg/mL was 68.5% for isolates that were OXA-1 positive and 93.8% for isolates that were OXA-1 negative.ConclusionThe MIC50 and MIC90 values of piperacillin-tazobactam among ESBL-producing E. coli were higher for the subset of isolates that harbored a narrow spectrum OXA-1 beta-lactamase enzyme relative to the subset that did not. This association was primarily observed among ESBL-producers with the CTX-M-15 enzyme variant. OXA-1 was infrequently detected among isolates with other ESBL enzyme types.Disclosures George Zhanel, PhD, AVIR (Grant/Research Support)Iterum (Grant/Research Support)Merck (Grant/Research Support)Pfizer (Grant/Research Support)Sandoz (Grant/Research Support)Sunovion (Grant/Research Support)Venatorx (Grant/Research Support)Verity (Grant/Research Support)

Significant contribution of the CmeABC Efflux pump in high-level resistance to ciprofloxacin and tetracycline in Campylobacter jejuni and Campylobacter coli clinical isolates

BackgroundCampylobacter resistance to antimicrobial agents is regarded as a major concern worldwide. The aim of this study was to investigate the expression of the CmeABC efflux pump and the RAPD-PCR pattern in drug-resistant Campylobacter isolates.MethodsA total of 283 stool specimens were collected from children under the age of five with diarrhea. The minimum inhibitory concentration (MIC) of tetracycline and ciprofloxacin was determined by broth microdilution method and E-test, respectively. Detection of tetracycline and ciprofloxacin determinants was done by amplification of tetO gene and PCR-sequencing of the gyrA gene. The cmeABC transcriptional expression was analyzed by Real-time (RT)-PCR. Clonal correlation of resistant strains was determined by RAPD-PCR genotyping.ResultsOut of 283 fecal samples, 20 (7.02%) samples were positive for Campylobacter spp. Analysis of duplex PCR assay of the cadF gene showed that 737 and 461 bp amplicons were corresponding to Campylobacter jejuni and Campylobacter coli, respectively. All of the 17 phenotypically tetracycline-resistant Campylobacter isolates harbored the tetO gene. Also, four phenotypically ciprofloxacin-resistant Campylobacter isolates had a point mutation at codon 257 of the gyrA gene (ACA to ATA; Thr > Ile). High-level expression of the cmeA gene was observed in ciprofloxacin-resistant and high-level tetracycline-resistant Campylobacter isolates, suggesting a positive correlation between the cmeA gene expression level and tetracycline resistance level. Moreover, a statistically significant difference was observed in the cmeA gene expression between ciprofloxacin-resistant and ciprofloxacin-susceptible strains, which signifies the crucial contribution of the efflux pump in conferring multiple drug resistance phenotype among Campylobacter spp. RAPD analysis of Campylobacter isolates exhibited 16 different patterns. Simpsone`s diversity index of RAPD-PCR was calculated as 0.85, showing a high level of homogeneity among the population; however, no clear correlation was detected among tetracycline and/or ciprofloxacin resistant isolates.ConclusionSignificant contribution of the CmeABC efflux pump in conferring high-level resistance to tetracycline and ciprofloxacin was observed in C. jejuni and C. coli clinical isolates. The resistant phenotype is suggested to be mediated by CmeABC efflux pumps, the tetO gene, and point mutation of the gyrA gene. Genotyping revealed no clonal correlation among resistant strains, indicating distinct evolution of tetracycline and ciprofloxacin resistant genotypes among the isolates.

Exploring the Contribution of the AcrB Homolog MdtF to Drug Resistance and Dye Efflux in a Multidrug Resistant E. coli Isolate.

In Escherichia coli, the role of RND-type drug transporters other than the major efflux pump AcrB has largely remained undeciphered (particularly in multidrug resistant pathogens), because genetic engineering in such isolates is challenging. The present study aimed to explore the capability of the AcrB homolog MdtF to contribute to the extrusion of noxious compounds and to multidrug resistance in an E. coli clinical isolate with demonstrated expression of this efflux pump. An mdtF/acrB double-knockout was engineered, and susceptibility changes with drugs from various classes were determined in comparison to the parental strain and its acrB and tolC single-knockout mutants. The potential of MdtF to participate in the export of agents with different physicochemical properties was additionally assessed using accumulation and real-time efflux assays with several fluorescent dyes. The results show that there was limited impact to the multidrug resistant phenotype in the tested E. coli strain, while the RND-type transporter remarkably contributes to the efflux of all tested dyes. This should be considered when evaluating the efflux phenotype of clinical isolates via dye accumulation assays. Furthermore, the promiscuity of MdtF should be taken into account when developing new antibiotic agents.

Real-Time Respiration Changes as a Viability Indicator for Rapid Antibiotic Susceptibility Testing in a Microfluidic Chamber Array.

Rapid identification of a pathogen and the measurement of its antibiotic susceptibility are key elements in the diagnostic process of bacterial infections. Microfluidic technologies offer great control over handling and manipulation of low sample volumes with the possibility to study microbial cultures on the single-cell level. Downscaling the dimensions of cultivation systems directly results in a lower number of bacteria required for antibiotic susceptibility testing (AST) and thus in a reduction of the time to result. The developed platform presented in this work allows the reading of pathogen resistance profiles within 2–3 h based on the changes of dissolved oxygen levels during bacterial cultivation. The platform contains hundreds of individual growth chambers prefilled with a hydrogel containing oxygen-sensing nanoprobes and different concentrations of antibiotic compounds. The performance of the developed platform is tested using quality control Escherichia coli strains (ATCC 25922 and ATCC 35218) in response to clinically relevant antibiotics. The results are in agreement with values given in reference guidelines and independent measurements using a clinical AST protocol. Finally, the platform is successfully used for the AST of an E. coli clinical isolate obtained from a patient blood culture.

Diverse and emerging molecular mechanisms award polymyxins resistance to Enterobacteriaceae clinical isolates from a tertiary hospital of Recife, Brazil.

To describe the molecular mechanisms of polymyxins resistance in five Enterobacteriaceae clinical isolates from a tertiary hospital of Recife, Brazil. The species identification and the susceptibility to antimicrobials were firstly performed by automatized methods and polymyxin resistance was confirmed by broth microdilution methods. The genetic basis of resistance was characterized with WGS analyses to study their resistome, plasmidome and mobilome, by BLAST searches on reference databases. Five (5%) Enterobacteriaceae isolates, comprising Escherichia coli (n=2), Klebsiella pneumoniae (n=2) and Citrobacter freundii (n=1) species, exhibited polymyxin resistance. The mcr-1.1 gene was found in identical IncX4-plasmids harbored by both K. pneumoniae C119 (PolB MIC=512mg/L) and E. coli C153 (PolB MIC=8mg/L). The remaining E. coli strain C027 harbored the mcr-5.1 gene on an undefined Inc-plasmid (PolB MIC 256mg/L). Some amino acid substitutions in PmrA (S29G, G144S), PmrB (S202P; D283G, W350*, Y258N) and PhoP (I44L) was detected among the E. coli clinical isolates, however they were also found in colistin-susceptible strains and predicted as neutral alterations. The mgrB of the ST54 KPC-2-producing K. pneumoniae C151 (PolB MIC=32g/mL) was interrupted at 69nt by the IS903 element. The ST117 C. freundii C156 (PolB MIC=256mg/L) showed the A91T substitution on HAMP domain of the histidine kinase sensor CrrB, predicted as deleterious and deemed the remarkable determinant to polymyxins resistance in this strain. Diverse mechanisms of polymyxins resistance were identified among clinical Enterobacteriaceae from a tertiary hospital of Recife, Brazil, such as plasmid-mediated MCR-1 and MCR-5; IS903-interruption of mgrB and mutation in CrrAB regulatory system. These findings highlight the involvement of the identified plasmids on mcr dissemination among Enterobacteriaceae; warn about co-selection of the polymyxin-resistant and KPC-producer K. pneumoniae ΔmgrB lineage by carbapenems usage; and demonstrate potential role of CrrAB on emerging of polymyxin resistance among Enterobacteriaceae, besides Klebsiella species.

Antibacterial and Antifungal Activities of the Leaf Exudate of Aloe megalacantha Baker.

Infectious diseases caused by fungi and bacteria are among the major causes of illness and death worldwide. This is mainly implicated by the antimicrobial resistance of the current treatment regimens. Since plant products are house stores of bioactive compounds, it is essential to screen plant-based antimicrobials to come up with novel medicines that counter the grave consequences of antimicrobial resistance. In the folk medicine of Ethiopia, Aloe megalacantha is used for the treatment of wound, dandruff, malaria, diabetes, impotence, colon cleansing, amoeba, ascariasis, abdominal pain, urine retention, snake bite, and evil eye. Hence, the present study was aimed to evaluate the antibacterial and antifungal effects of the leaf exudate of Aloe megalacantha. Agar well diffusion was employed to determine the antibacterial and antifungal effects. Six bacterial strains, namely, S. aureus (standard), S. aureus (clinical isolate), E. coli ATCC 25922 (standard), E. coli (clinical isolate), K. pneumoniae (standard), and P. aeruginosa ATCC 27853 (standard), and four fungal strains such as C. albicans, C. glabrata, C. tropicalis, and C. krusei were studied. The leaf exudate showed the highest activity against C. krusei with an average zone diameter of 22.49 ± 0.47 mm at 400 mg/mL. Among the bacterial species, S. aureus ATCC 29213 (standard) was the most sensitive with an average zone of diameter of 16.63 ± 0.12 mm at 200 mg/mL. Thus, the present findings support the folklore use of Aloe megalacantha for the treatment of different microbial infections.

550. Carbapenem--Resistant E. coli and A. baumannii Among Catheter-Related Blood Stream Infection Patients in Egyptian ICUs

BackgroundThe spreading of E. coli and A. baumannii in hospitals is a growing concern due to increased resistance to carbapenems and Fluoroquinolones. The present study aimed to specifically evaluate the presence of mutations in the gyrA and parC genes in Egyptian ICU and their correlation with carbapenem-resistant genes E. coli and A. baumannii isolates from patients in 4 tertiary care hospital in Egypt.MethodsA total of 120 A. baumannii and E. coli clinical isolates were isolated from ICU patients in 4 tertiary hospitals in Egypt. The bacterial isolates were identified by VITEK-2 (Bio Merieux, France). Antimicrobial susceptibility testing was performed according to CLSI guidelines. Phenotypic detection of carbapenemase activity was done by carba-NP test, followed by molecular identification of carbapenemase encoding genes blaNDM, blaOXA-48 and blaKPC by multiplex PCR. The quinolone resistance-determining regions (QRDRs) of gyrA and parC genes were amplified by singleplex PCR followed by reverse and forward sequencing to detect the gene mutation. The DNA sequences were compared with the sequences of wild type of these genes available in GenBank database. Then, the obtained DNA sequences and their amino acid sequences were analyzed using bioinformatics tools.Results All isolates showed a high level of resistance among tested antimicrobial agents (cephalosporins, aminoglycosides, carbapenems, penicillins) that ranged from 36% to 100%. Carba-NP detected 43.59% of the carbapenem-resistant isolates. Multiplex PCR detected that 17.95%, 46.15% and 2.56% of isolates were harboring blaKPC, blaNDM and blaOXA-48 respectively. PCR and sequencing technique showed combined gene mutation in 8 carbapenem-resistant E. coli and A. baumannii isolates. The specific substitutions observed in gyrA were Cys173Arg, Cys174 Gly, Asp80Val, Tyr178ASP, Tyr84Gly, Glu85Lys, Ser172Leu and Asp176Asn. While, the specific substitutions observed in parC were point mutation 62 Arg, Phe60Leu, Ils66Val, Gln76Lys. Point mutation 62 Arg was observed in two A. baumannii isolates, whereas Ser172Leu mutation was observed in two E. coli isolates.ConclusionThe presence of carbapenem resistance genes in combination with single and multiple mutations in QRDR causes the presence of highly resistant E. coli and A. baumannii isolates in the Egyptian hospitals.Disclosures All authors: No reported disclosures.

2609. Escherichia coli Clonal Lineages and Virulence Factors Predict Fecal Colonization within Households

BackgroundExtraintestinal Escherichia coli infections are an ever-growing threat, to which specific clonal lineages and virulence factors contribute disproportionately. Despite the gut being the main reservoir for such E. coli strains, relationships between clonal lineages, virulence factors, and fecal colonization patterns are poorly understood. Accordingly, we defined E. coli fecal colonization patterns within households (HHs) and assessed specific lineages and virulence genes (VGs) as predictors of colonization behaviors.MethodsVeterans with an E. coli clinical isolate (n = 22: 11 fluoroquinoline [FQ]-resistant, 11 FQ-susceptible) and their HH members provided stool samples on 2–6 occasions each. Stools were screened for total and FQ-resistant E. coli. Distinct E. coli strains were resolved by genomic profiling of 10 colonies/sample. Strains underwent molecular lineage identification, VG detection, and comparison with the veteran’s clinical isolate. Clonal lineages and VGs were assessed (Wilcoxon rank-sum test) as predictors of strains’ (i) predominance within the fecal sample, (ii) persistence across serial fecal samples, (iii) within-HH strain sharing, and (iv) overall within-HH colonization prevalence.ResultsFrom the 22 veterans and 46 HH members (27 humans, 19 pets) we recovered 139 unique-by-household fecal E. coli strains. Sixty-four traits were evaluated (16 clonal lineages, 48 VGs). Of these, 44 exhibited n ≥ 5, so could be analyzed statistically. Among these 44 traits, the proportion significantly associated with ≥ 1 outcome variable was 5/6 (83%) for clonal lineages and 18/38 (47%) for VGs. Additionally, fecal strains that matched the veteran’s clinical isolate exhibited significantly greater sharing, persistence, and overall colonization.ConclusionThe studied E. coli traits – known for their associations with clinical infections –here were significantly associated with within-HH colonization behavior. These findings support that “virulence factors” may be regarded also (or perhaps best) as “colonization factors,” and “virulent lineages” as “colonizing lineages.” This suggests the possibility that future interventions that disrupt colonization behavior also could prevent E. coli infections. Disclosures All authors: No reported disclosures.