Are Uropathogenic Bacteria Living in Multispecies Biofilm Susceptible to Active Plant Ingredient-Asiatic Acid?

Urinary tract infections (UTIs) are a serious health problem in the human population due to their chronic and recurrent nature. Bacteria causing UTIs form multispecies biofilms being resistant to the activity of the conventionally used antibiotics. Therefore, compounds of plant origin are currently being searched for, which could constitute an alternative strategy to antibiotic therapy. Our study aimed to determine the activity of asiatic acid (AA) against biofilms formed by uropathogenic Escherichia coli, Enterobacter cloacae, and Pseudomonas aeruginosa. The influence of AA on the survival, biofilm mass formation by bacteria living in mono-, dual-, and triple-species consortia as well as the metabolic activity and bacterial cell morphology were determined. The spectrophotometric methods were used for biofilm mass synthesis and metabolic activity determination. The survival of bacteria was established using the serial dilution assay. The decrease in survival and a weakening of the ability to create biofilms, both single and multi-species, as well as changes in the morphology of bacterial cells were noticed. As AA works best against young biofilms, the use of AA-containing formulations, especially during the initial stages of infection, seems to be reasonable. However, there is a need for further research concerning AA especially regarding its antibacterial mechanisms of action.

Norovirus diarrhea is significantly associated with higher counts of fecal histo-blood group antigen expressing Enterobacter cloacae among black South African infants.

The study tested the hypothesis that harboring high levels of histo-blood group antigen-expressing Enerobactero cloacae is a risk factor for norovirus diarrhea. The fecal E. cloacae abundance in diarrheic norovirus positive (DNP), non-diarrheic norovirus negative (NDNN), diarrhea norovirus negative (DNN), and non-diarrhea norovirus positive (NDNP) infants was determined by qPCR, and the risk of norovirus diarrhea was assessed by logistical regression. DNP infants contained significantly higher counts of E. cloacae than NDNN and DNN infants, p = .0294, and 0.0001, respectively. The risk of norovirus diarrhea was significantly high in infants with higher counts of E. cloacae than those with lower counts, p = .009. Harboring higher counts of E. cloacae is a risk factor for norovirus diarrhea.

Nettle-Leaf Extract Derived ZnO/CuO Nanoparticle-Biopolymer-Based Antioxidant and Antimicrobial Nanocomposite Packaging Films and Their Impact on Extending the Post-Harvest Shelf Life of Guava Fruit.

Green synthesized metal oxide nanoparticles (NPs) have prominent applications in antimicrobial packaging systems. Here we have attempted for the fabrication of chitosan-based nanocomposite film containing Urtica dioica leaf extract derived copper oxide (CuO) and zinc oxide (ZnO) NPs for shelf-life extension of the packaged guava fruits. Electron microscopy and spectroscopy analysis of the CuO and ZnO NPs exhibited nano-scale size, spherical morphologies, and negative ζ-potential values. The NPs possessed appreciable antioxidant and antimicrobial activity (AMA) in order of CuO NPs > ZnO NPs >nettle extract. Therefore, this work establishes for the first time the successful synthesis of CuO NPs and compares its antimicrobial and antioxidant properties with ZnO NPs. On incorporation in chitosan, the polymer nanocomposite films were developed by solvent casting technique. The developed films were transparent, had low antioxidant but substantial AMA. The NP supplementation improved the film characteristics as evident from the decrease in moisture content, water holding capacity, and solubility of the films. The nanocomposite films improved the quality attributes and shelf life of guava fruits by one week on packaging and storage compared to unpackaged control fruits. Therefore, this study demonstrates the higher antimicrobial potential of the nettle leaf extract derived CuO/ZnO NPs for development of antimicrobial nanocomposite films as a promising packaging solution for enhancing the shelf life of various perishable fruits.

Coexistence of Fosfomycin Resistance Determinant fosA and fosA3 in Enterobacter cloacae Isolated from Pets with Urinary Tract Infection in Taiwan.

To analyze the characteristics of fosA and fosA3 in Enterobacter cloacae isolated from aspirated and catheterized urine culture specimens of companion pets in Taiwan. A total of 19 E. cloacae isolates from pets with urinary tract infection were screened for the presence of fosA, fosA3, and fosC2 and for the genetic context of them by PCR amplification and sequencing. The transferability, resistance phenotypes, plasmid replicon typing properties and genetic environments of fosA- and/or fosA3-positive strains were characterized. Five E. cloacae isolates were positive for fosA and three coharbored fosA and fosA3. No fosC determinant was detected. Transconjugants of fosA3 were successfully acquired, while the acquisition of fosA transconjugants was failed. The minimum inhibitory concentrations (MICs) of the three fosA3-positive isolates and their transconjugants were ≥256 mg/L, whereas the MICs of the five fosA-positive isolates ranged from 64 mg/L to 256 mg/L. Three plasmid replicons (InCFrepB, InCL/M, and InCHI2) were identified in fosA- and fosA3-positive E. cloacae isolates. Different genetic contexts lay in the downstream region of fosA and fosA3, respectively. Eight distinct patterns based on the similarity value of more than 80% were typed for all the 8 fosA-positive isolates. In conclusion, the fosA concomitant with fosA3 were found in E. cloacae isolates. The fosA3 not only exhibits stronger activity of inactivating fosfomycin than fosA but also possesses stronger potential to spread than fosA. Different genetic backgrounds exist in these fosA- and fosA3-positive isolates, and different mobile elements may confer the dissemination of fosA and fosA3.

Impact of Antibiotic-Resistant Bacteria on Immune Activation and Clostridioides difficile Infection in the Mouse Intestine.

Antibiotic treatment of patients undergoing complex medical treatments can deplete commensal bacterial strains from the intestinal microbiota, thereby reducing colonization resistance against a wide range of antibiotic-resistant pathogens. Loss of colonization resistance can lead to marked expansion of vancomycin-resistant Enterococcus faecium (VRE), Klebsiella pneumoniae, and Escherichia coli in the intestinal lumen, predisposing patients to bloodstream invasion and sepsis. The impact of intestinal domination by these antibiotic-resistant pathogens on mucosal immune defenses and epithelial and mucin-mediated barrier integrity is unclear. We used a mouse model to study the impact of intestinal domination by antibiotic-resistant bacterial species and strains on the colonic mucosa. Intestinal colonization with K. pneumoniae, Proteus mirabilis, or Enterobacter cloacae promoted greater recruitment of neutrophils to the colonic mucosa. To test the hypothesis that the residual microbiota influences the severity of colitis caused by infection with Clostridioides difficile, we coinfected mice that were colonized with ampicillin-resistant bacteria with a virulent strain of C. difficile and monitored colonization and pathogenesis. Despite the compositional differences in the gut microbiota, the severity of C. difficile infection (CDI) and mortality did not differ significantly between mice colonized with different ampicillin-resistant bacterial species. Our results suggest that the virulence mechanisms enabling CDI and epithelial destruction outweigh the relatively minor impact of less-virulent antibiotic-resistant intestinal bacteria on the outcome of CDI.

Improved Biosurfactant Production by Enterobacter cloacae B14, Stability Studies, and its Antimicrobial Activity.

This work aimed to optimize carbon and nitrogen sources for the growth of Enterobacter cloacae B14 and its biosurfactant (BS) production via One-Variable-At-a-Time (OVAT) method. The BS stability under a range of pH and temperatures was assessed. Antimicrobial activity against Gram-positive and Gram-negative pathogens was determined by the agar well diffusion method. The results showed that the optimum carbon and nitrogen sources for BS production were maltose and yeast extract, respectively, with a maximum BS yield of (39.8 ± 5.2) mg BS/g biomass. The highest emulsification activity (E24) was 79%, which is significantly higher than in the previous studies. We found that B14 BS can withstand a wide range of pH values from 2 to10. It could also function under a range of temperatures from 30-37°C. Thin Layer Chromatography (TLC) and Fourier Transform Infrared Spectrometry (FTIR) analysis confirmed that B14 BS is a glycolipid-like compound, which is rarely found in Enterobacter spp. Cell-free broth showed inhibition against various pathogens, preferable to Gram-positive ones. It had better antimicrobial activity against Bacillus subtilis than a commonly-used antibiotic, tetracycline. Furthermore, B14 broth could inhibit the growth of a tetracycline-resistant Serratia marcescens. Our results showed promising B14 BS applications not only for bioremediation but also for the production of antimicrobial products.

Characterization of Carbapenem-Resistant Enterobacteriaceae Clinical Isolates in Al Thawra University Hospital, Sana'a, Yemen.

Objective: The aim of this study was to investigate the resistance mechanisms of carbapenem-resistant Enterobacteriaceae clinical strains recovered from Al Thawra University Hospital, Sana'a, Yemen. Methods: A total of 27 isolates showing decreased susceptibility to carbapenems were obtained from different clinical specimens in Al Thawra Hospital, Sana'a, Yemen. Strains were identified by Matrix Assisted Laser Desorption Ionization Time-Of-Flight spectroscopy. Susceptibility to antibiotics was determined by the disk diffusion method on Mueller Hinton agar. Carbapenemases-encoding genes, extended-spectrum ß-lactamases (ESBLs), and plasmid-mediated quinolone resistance (PMQR) genes were screened by PCR. Bacterial isolates were typed by multilocus sequence typing (MLST). Results: Carbapenemase genes detection and sequencing showed that 18 (66.7%) isolates were Klebsiella pneumoniae (NDM-1, n = 13; NDM-1 + OXA-48, n = 3; OXA-48, n = 1; OXA-232, n = 1), 6 (22.2%) were Escherichia coli (NDM-5, n = 3; OXA-181, n = 2; OXA-48, n = 1), and 3 (11.1%) were Enterobacter cloacae (NDM-1, n = 1; OXA-181, n = 2). In addition the ESBL gene blaCTX-M-15 was detected in 14 K. pneumoniae and 2 E. coli isolates, and the blaCTX-M-216 was found in 1 E. coli isolate. Fifteen isolates were PMQR positive including qnrB1 (n = 1), qnrS1 (n = 5), qnrS4 (n = 2), and aac-(6')-Ib-cr (n = 7). The MLST typing showed a diversity of sequence type (ST) clones including Escherichia coli ST410 (3), ST448 (2), and ST648; Enterobacter cloacae ST78 and ST270; and Klebsiella pneumoniae ST395 (2), ST309, ST23, ST35, ST1728, ST15, ST231, and ST1428. Conclusion: This study reports the first description of OXA-48-like-producing Enterobacteriaceae and NDM-5 enzymes in E. coli in Yemen.

Model-Informed Drug Development, Pharmacokinetic/Pharmacodynamic Cutoff Value Determination, and Antibacterial Efficacy of Benapenem against Enterobacteriaceae.

Benapenem is a novel carbapenem. The objective of this study was to determine the pharmacokinetic (PK)/pharmacodynamic (PD) cutoff values and evaluate the optimal administration regimens of benapenem for the treatment of bacterial infections via PK/PD modeling and simulation. Ertapenem was used as a control. Infected mice received an intravenous (i.v.) injection of benapenem or ertapenem of 14.6, 58.4, or 233.6 mg/kg of body weight, and the PK/PD profiles were evaluated. The MICs were determined by using a 2-fold agar dilution method. Mathematical models were developed to characterize the pharmacokinetic profile of benapenem in humans and mice. Monte Carlo simulations were employed to determine the cutoff values and the appropriate benapenem dosing regimens for the treatment of infections caused by clinical isolates of Enterobacteriaceae Two 2-compartment models were developed to describe the PK profiles of benapenem in humans and mice. A two-site binding model was applied to fit the protein binding in mouse plasma. Through correlation analysis, the percentage of the time that the free drug concentration remains above the MIC (%fT>MIC) was determined to be the indicator of efficacy. Results from the simulation showed that the probability of target attainment (PTA) against the tested isolates was over 90% with the dosing regimens studied. The PK/PD cutoff value of benapenem was 1 mg/liter at a %fT>MIC of 60% when given at a dose of 1,000 mg/day by i.v. drip for 0.5 h. The established model provides a better understanding of the pharmacological properties of benapenem for the treatment of Enterobacteriaceae infections. The proposed PK/PD cutoff value suggests that benapenem is a promising antibacterial against the Enterobacteriaceae The cutoff value of 1 mg/liter may be a useful guide for the clinical use of benapenem and for surveillance for benapenem resistance.

Spheroplast-Mediated Carbapenem Tolerance in Gram-Negative Pathogens.

Antibiotic tolerance, the ability to temporarily sustain viability in the presence of bactericidal antibiotics, constitutes an understudied and yet potentially widespread cause of antibiotic treatment failure. We have previously shown that the Gram-negative pathogen Vibrio cholerae can tolerate exposure to the typically bactericidal ß-lactam antibiotics by assuming a spherical morphotype devoid of detectable cell wall material. However, it is unclear how widespread ß-lactam tolerance is. Here, we tested a panel of clinically significant Gram-negative pathogens for their response to the potent, broad-spectrum carbapenem antibiotic meropenem. We show that clinical isolates of Enterobacter cloacae, Klebsiella aerogenes, and Klebsiella pneumoniae, but not Escherichia coli, exhibited moderate to high levels of tolerance of meropenem, both in laboratory growth medium and in human serum. Importantly, tolerance was mediated by cell wall-deficient spheroplasts, which readily recovered wild-type morphology and growth upon removal of antibiotic. Our results suggest that carbapenem tolerance is prevalent in clinically significant bacterial species, and we suggest that this could contribute to treatment failure associated with these organisms.

Role of coaggregation in the pathogenicity and prolonged colonisation of Vibrio cholerae.

Cholera is an acute diarrheal illness caused by the Gram-negative bacterium Vibrio cholerae. The pathogen is known for its ability to form biofilm that confers protection against harsh environmental condition and as part of the colonisation process during infection. Coaggregation is a process that facilitates the formation of biofilm. In a preliminary in vitro study, high coaggregation index and biofilm production were found between V. cholerae with human commensals namely Escherichia coli and Enterobacter cloacae. Building upon these results, the effects of coaggregation were further evaluated using adult BALB/c mouse model. The animal study showed no significant differences in mortality and fluid accumulation ratio between treatment groups infected with V. cholerae alone and those infected with coaggregation partnership (V. cholerae with E. coli or V. cholerae with E. cloacae). However, mild inflammation was detected in both partnering pairs. Higher density of V. cholerae was recovered from faecal samples of mice co-infected with E. coli and V. cholerae in comparison with other groups at 24 h post-infection. This partnership also elicited slightly higher levels of interleukin-5 (IL-5) and interleukin-10 (IL-10). Nonetheless, the involvement of autoinducer-2 (AI-2) as the signalling molecules in quorum sensing system is not evident in this study. Since E. coli is one of the common commensals, our result may suggest the involvement of commensals in cholera development.

Antibacterial and antidiarrheal activity of Butea Monospermea bark extract against waterborne enterobacter Cloacae in rodents: In-vitro, Ex-vivo and In-Vivo evidences.

ETHNOPHARMACOLOGICAL RELEVANCE: Butea monosperma (Lam.) Taub. (family Leguminosae), popularly known as 'Palash' possess numerous medicinal properties since ancient times. According to the Wealth of India, stem bark of this plant exhibits various therapeutic properties like antimicrobial, astringent, styptic, aphrodisiac, and anti-inflammatory. AIM OF THE STUDY: The purpose of the present study was to investigate antibacterial and antidiarrheal effect of B. monosperma bark against newly isolated gram negative pathogenic bacterial strain Enterobacter cloacae. MATERIALS AND METHODS: Aqueous extract of B. monosperma bark (BMAqE) was subjected to LC-MS/MS analysis for determination of bioactive components. Antibacterial study of BMAqE was assessed using bacterial growth kinetic study, fluorescence spectroscopy, outer and inner membrane permeability assay, dehydrogenase inhibitory assay and protein leakage assay followed by field emission scanning electron microscope (FE-SEM) study. Antidiarrheal activity was studied using castor oil induced diarrhea model in albino rats followed by histopathology studies of rat ileum. RESULTS: LC-MS/MS analysis of BMAqE revealed presence of twenty-two different active phytoconstituents out of which most of the constituents belong to flavonoid and polyphenol family. BMAqE showed MIC and MBC (IC90) value of 5 and 200 µg/mL against targeted bacterial strain. BMAqE exhibited potent and dose dependent bactericidal effect via disruption of integrity of bacterial cell membrane, enzymatic degradation, leakage of intracellular protein and ruptured bacterial cell. In castor oil induced diarrhea model, BMAqE (200 mg/kg; orally) caused marked reduction (75.66%) in the frequency of defecation and mean weight of faeces (0.54 ±â€¯0.04) when compared to control group (2.26 ±â€¯0.25). Histopathology study revealed marked restoration of cellular architecture of rat ileum tissue. Four known flavonoids were isolated from BMAqE using column chromatography. In ex-vivo study, BMAqE (0.0002, 0.0004 and 0.0006 g/L) and isolated flavonoids i.e. rhamnetin, quercetin, kaempferol and catechin (0.5, 5 & 50 µm) produced a significant (p < 0.001) change in EC50 and indicated competitive phenomena via rightward shift of acetylcholine CRC with pA2 of 3.78, 8.0, 7.1, 7.0 and 6.9 respectively. CONCLUSION: BMAqE exhibits impressive antibacterial and anti-diarrheal activity and can be effectively used to eradicate water borne diseases.

Co-culture with Enterobacter cloacae does not Enhance Virus Resistance to Thermal and Chemical Treatments.

Human noroviruses (hNoV) are the primary cause of foodborne disease in the USA. Most studies on inactivation kinetics of hNoV and its surrogates are performed in monoculture, while the microbial ecosystem effect on virus inactivation remains limited. This study investigated the persistence of hNoV surrogates, murine norovirus (MNV) and Tulane virus (TuV), along with Aichi virus (AiV) under thermal and chemical inactivation in association with Gram-negative (Enterobacter cloacae) bacteria. Thermal inactivation of viruses in co-culture with E. cloacae revealed no protective effects of bacteria. At 56 °C, AiV with and without bacteria was completely inactivated by 10 min with decimal reduction values (D-values) of 41 and 43 s, respectively. Similar results were also observed for TuV. Conversely, MNV with bacteria was completely inactivated by 10 min while MNV alone remained stable up to 30 min at 56 °C. Both MNV and TuV were slightly more stable than AiV at 63 °C with TuV detection up to 2 min without bacteria. For chemical inactivation on stainless steel surfaces, viruses alone and in association with bacteria were treated with 1000 ppm sodium hypochlorite. Virus association with bacteria had no significant effect (p > 0.05) on virus resistance to bleach inactivation compared to virus alone. Specifically, exposure to 1000 ppm bleach for 5 min resulted in an average of 3.86, 2.14, and 0.94 log10 PFU/ml reductions for TuV, MNV, and AiV without bacteria, respectively. Reductions in TuV, MNV, and AiV were 3.50, 1.88, and 0.61 log10 PFU/ml when associated with E. cloacae, respectively.

Impact of plant growth promoting rhizobacteria on different forms of soil potassium under wheat cultivation.

This study aimed to investigate the effect of some plant growth promoting rhizobacteria with potassium dissolution ability on different forms of potassium in soil under the cultivation of wheat. The factorial experiment was conducted as a randomized complete design with three replications in greenhouse conditions. The treatments consisted of bacterium inoculation (without inoculation, Enterobacter cloacae Rhizo_33, Pseudomonas sp. Rhizo_9, consortium of Enterobacter cloacae Rhizo_33 and Pseudomonas sp. Rhizo_9), potassium application (2·87 mg K kg-1 of soil and without potassium application).The results indicated that soils treated with Enterobacter cloacae Rhizo_33, either receiving potassium or not, maintain a higher amount of exchangeable K (337 mg kg-1 ) and water-soluble K (1·25 and 1·31 meq L-1 with and without K application respectively). The nonexchangeable K and nitric acid-extractable K values were decreased by inoculating bacterial strains. The grain yield was significantly enhanced by the inoculation of bacterial strains irrespective of rates of potassium application. About 19·16% increase of grain yield was recorded by inoculation of Enterobacter cloacae Rhizo_33 and without potassium application. A significantly greater amount of K uptake in grain was obtained in soils treated with Enterobacter cloacae Rhizo_33, with and without the application of potassium (28·7 and 30·7 mg per pot respectively). There was a significant (P < 0·01) and positive correlation between grain yield and grain, shoot and root K uptake. Potassium uptake had a positive significant correlation with water-soluble K and exchangeable K; it was negatively correlated with K (HNO3 ). The data suggested that inoculation of soil with mentioned bacteria can improve plant growth and potassium uptake. SIGNIFICANCE AND IMPACT OF THE STUDY: As one of the macronutrients, Potassium is the most abundant absorbed cation in most plants and exists in soil in different forms. Soluble and exchangeable forms of potassium (K) are important with regard to plant uptake. K-solubilizing bacteria can convert insoluble potassium to soluble forms; therefore using K-solubilizing bacteria as biofertilizers is a sustainable solution for the improvement of plant growth, nutrition, root growth, plant competitiveness and reducing the use of potassium chemical fertilizer.

Single-cell variability of growth interactions within a two-species bacterial community.

Cell-cell interaction networks have been examined in many high diversity microbial communities using macroscale approaches. Microscale studies of multispecies communities are lacking and it remains unclear how macroscale trends scale down to small groups of cells. Experimental approaches using microfluidic devices have revealed heterogeneity in the behavior of single cells, however, this analysis has not been extended towards the variability of cell-cell interactions. Using a microwell device, we analyzed cell growth within hundreds of replicate microbial communities consisting of two species and small population sizes. The wells of the devices were inoculated with a coculture of Escherichia coli and Enterobacter cloacae. Each species expressed a unique fluorescent protein enabling simultaneously tracking of cell number for each species over time. Growth dynamics within the device were consistent with bulk measurements. The device enabled monitoring of replicate, isolated coculture populations at high magnification, revealing both the growth interaction between the two species and the variability of such cell-cell interactions within small groups of cells. The device enables new experimental measurements of the heterogeneity of interactions within small, multispecies populations of bacteria.

Sequencing of pT5282-CTXM, p13190-KPC and p30860-NR, and comparative genomics analysis of IncX8 plasmids.

This study proposes a replicon-based scheme for typing IncX plasmids into nine separately clustering subgroups, including IncX1α, IncX1ß and IncX2-8. The complete nucleotide sequences of three IncX8 plasmids, namely pT5282-CTXM and p30860-NR from Enterobacter cloacae and p13190-KPC from Klebsiella pneumoniae, were determined and were compared with two other previously sequenced IncX8 plasmids (pCAV1043-58 and pCAV1741-16). These five plasmids possessed conserved IncX8 backbones with limited genetic variation with respect to gene content and organisation, and each of them carried one or three accessory modules that harboured resistance markers and metabolic gene clusters as well as transposons, insertion sequence (IS)-based transposition units and miniature inverted repeat transposable elements (MITEs), indicating that the relatively small IncX8 backbones were able to integrate various foreign genetic contents. The resistance genes blaCTX-M-3 and blaTEM-1 (ß-lactam resistance), blaKPC-2 (carbapenem resistance) and ΔblaTEM-1, and tet(A) (tetracycline resistance) and mph(E) (macrolide resistance) were found in pT5282-CTXM, p13190-KPC and pCAV1741-16, respectively, whilst p30860-NR and pCAV1043-58 carried no resistance genes. The data presented here provide an insight into the diversification and evolution history of IncX8 plasmids.

Evaluation of the rapid carbapenem inactivation method (rCIM): a phenotypic screening test for carbapenemase-producing Enterobacteriaceae.

Objectives: Fast and accurate diagnostic tests to identify carbapenemase-producing Enterobacteriaceae (CPE) are mandatory for proper antimicrobial therapy and implementing infection control measures. Here, we have developed a rapid Carbapenem Inactivation Method (rCIM) for CPE detection. Methods: The rCIM consists of the incubation of a potential carbapenemase producer with meropenem discs and use of the resulting supernatant to challenge a susceptible indicator strain. Growth of the indicator strain is monitored using a nephelometer. The performances of the rCIM were compared with the CIM and Carba NP tests using a collection of 113 well-characterized carbapenem-resistant enterobacterial isolates, including 85 carbapenemase producers and 28 non-carbapenemase producers. In addition, rCIM was compared with the Carba NP test and PCR sequencing in a prospective analysis of 101 carbapenem-resistant enterobacterial isolates addressed to the French National Reference Center for Antimicrobial Resistance in July 2017. Results and discussion: The rCIM correctly identified 84/85 carbapenemase producers and 28/28 non-carbapenemase producers, yielding a sensitivity of 99% and a specificity of 100%, slightly higher than the CIM and Carba NP test. In the prospective validation study, the rCIM showed a sensitivity and specificity of 97% and 95%, respectively. Two cephalosporinase-hyperproducing Enterobacter cloacae gave false-positive results, whereas an IMI-17-producing Enterobacter asburiae gave a false-negative result. The result was, however, positive when the isolate was grown on selective antibiotic-containing media. Conclusions: The rCIM is a rapid (less than 3 h), cheap and accurate test for the detection of CPEs, which can be implemented in low-resource settings, making it a useful tool for microbiology laboratories.

Antibiotic resistance in Enterobacter cloacae strains with derepressed/partly derepressed/inducible AmpC and extendedspectrum beta-lactamases in Zenica-Doboj Canton, Bosnia and Herzegovina.

Aim To investigate the prevalence of derepressed/partly derepressed/inducible and ESBL/AmpC-producing Enterobacter cloacae isolates and treatment options for infections associated with those isolates. Methods Antibiotic susceptibility was determined by disc diffusion and broth microdilution according to CLSI guidelines. Doubledisk synergy test (DDST) was performed in order to screen for ESBLs and combined disk test with phenylboronic acid to detect AmpC ß -lactamases. PCR was used to detect blaESBL/blacarb genes. Genetic relatedness of the strains was determined by pulsed-fieldgel-electrophoresis (PFGE). Results Among 14 isolates with the ESBL positive E. cloaceae producing isolates, four (28.6%), nine (64.3%) and one (7.1%) isolates were derepressed/partly derepressed and inducible AmpC producers. Eleven (out of 14) isolates were resistant to cefotaxime, ceftazidime, ceftriaxone, aminoglycosides and fluoroquinolones. All isolates were susceptible to imipenem and meropenem, 79% to cefepime. Five (out of 14; 35.7%) isolates (four derepressed and one inducible AmpC carrying E. cloaceae) were negative in phenotypic test for ESBLs, but positive for broad spectrum TEM-1 ß-lactamase. One (out of four derepressed) also produced CMY-2 ß-lactamase. Four (out of nine) partly derepressed isolates were positive with the DDST, but did not yield PCR products with primers targeting TEM, SHV and CTX-M beta-lactamases. Four positive partly derepressed isolates carried a blaCTX-M-1 gene, two blaOXA-1 one blaCTX-M-15, OXA-1 and one blaCTX-M-28, OXA-1 (n=1). Conclusion Microbiology laboratories must be able to detect and recognize AmpC-carrying isolates in a timely manner, especially those that are falsely susceptible in vitro to drugs that may be consideredfor therapy of infected patients.

Inhibition of Fosfomycin Resistance Protein FosA by Phosphonoformate (Foscarnet) in Multidrug-Resistant Gram-Negative Pathogens.

FosA proteins confer fosfomycin resistance to Gram-negative pathogens via glutathione-mediated modification of the antibiotic. In this study, we assessed whether inhibition of FosA by sodium phosphonoformate (PPF) (foscarnet), a clinically approved antiviral agent, would reverse fosfomycin resistance in representative Gram-negative pathogens. The inhibitory activity of PPF against purified recombinant FosA from Escherichia coli (FosA3), Klebsiella pneumoniae (FosAKP), Enterobacter cloacae (FosAEC), and Pseudomonas aeruginosa (FosAPA) was determined by steady-state kinetic measurements. The antibacterial activity of PPF against FosA in clinical strains of these species was evaluated by susceptibility testing and time-kill assays. PPF increased the Michaelis constant (Km ) for fosfomycin in a dose-dependent manner, without affecting the maximum rate (Vmax) of the reaction, for all four FosA enzymes tested, indicating a competitive mechanism of inhibition. Inhibitory constant (Ki ) values were 22.6, 35.8, 24.4, and 56.3 µM for FosAKP, FosAEC, FosAPA, and FosA3, respectively. Addition of clinically achievable concentrations of PPF (∼667 µM) reduced the fosfomycin MICs by ≥4-fold among 52% of the K. pneumoniae, E. cloacae, and P. aeruginosa clinical strains tested and led to a bacteriostatic or bactericidal effect in time-kill assays among representative strains. PPF inhibits FosA activity across Gram-negative species and can potentiate fosfomycin activity against the majority of strains with chromosomally encoded fosA These data suggest that PPF may be repurposed as an adjuvant for fosfomycin to treat infections caused by some FosA-producing, multidrug-resistant, Gram-negative pathogens.

Microbial decolorization and detoxification of emerging environmental pollutant: Cosmetic hair dyes.

Since the usage of hair dyes has increased in recent time, the removal of residual dye from environment is also an emerging issue. Hair dye contains mixture of chemicals including genotoxic chemical, p-phenylenediamine (p-PD or PPD). The present study reports bioremediation of hair dye using bacteria isolated from saloon effluent. Sugarcane bagasse powder (SBP) was used as a source of nutrient and surface for bacterial growth. The 16S rDNA sequencing confirmed the isolate as Enterobacter cloacae which was designated as DDB I. The decolourization of dye was studied using UV-vis spectrophotometer. The detoxification study was conducted on microbes isolated from fresh ponds using well diffusion assay. The 1mg/ml of dye was effectively decolourised within 18h of DDB I treatment in the minimal medium containing 30mg/ml of SBP.

Successful conservative management of a permanent pacemaker pocket infection: a less invasive approach.

We present a successful conservative management strategy for a frail elderly patient with a cardiac resynchronisation pacemaker who presented with evidence of an Enterobacter cloacae pacemaker pocket infection. A device washout and debridement procedure was performed, with reburial of the device in a new prepectoral pocket and creation of a closed-loop continuous antibiotic infusion into the infected pacemaker pocket. This was followed by a 6-week course of ambulatory intravenous antibiotic therapy. This conservative management strategy avoided the need for a more invasive and high-risk full device extraction, which the patient clearly stated he did not wish to have. Up-to-date consensus management guidelines recommend extraction of the entire implanted system in this situation; however, in this case we demonstrate an alternative conservative management option, which may be suitable for frail elderly and comorbid patients or for patients who decline device extraction.