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26.  Spread of Carbapenemase-producing Enterobacteria in a Southwest Hospital in China 
Background
The rapid emergence and dissemination of carbapenem resistance in Enterobacteriaceae complicates the treatment of infections caused by these organisms.
Methods
We collected clinical isolates with meropenem inhibition zones of ≤ 22 mm from January 1, 2009, through December 31, 2010. We attempted to amplify the NDM-1 gene from these isolates and conducted the modified Hodge test (MHT). The minimal inhibitory concentration (MIC) of the MHT-positive strains was determined by the agar disk dilution method. The carbapenemase-encoding resistance genes of these strains were examined using polymerase chain reaction (PCR) analysis and a sequencing strategy to characterize these enzymes. The clonal relationship among isolates was analyzed by pulsed-field gel electrophoresis (PFGE).
Results
Among the 158 Enterobacteriaceae isolates that were collected, there were no NDM-1-positive strains and 26 MHT-positive strains. Among the latter, 18 strains were IMP-4-positive, and 1 was KPC-2-positive. In addition, 15 of the IMP-4-positive Klebsiella pneumoniae strains belonged to 4 PFGE genotypes, with 8 strains having the same genotype.
Conclusion
These results suggest that nosocomial infections are one of the main reasons for the spread of these resistant strains.
doi:10.1186/s12941-014-0042-4
PMCID: PMC4236511  PMID: 25113057
Enterobacteria; Carbapenemase; Emergence; Spread; Resistance
27.  Purification of Chitinase enzymes from Bacillus subtilis bacteria TV-125, investigation of kinetic properties and antifungal activity against Fusarium culmorum 
Background
Chitin is the main structural component of cell walls of fungi, exoskeletons of insects and other arthropods and shells of crustaceans. Chitinase enzyme is capable of degrading chitin, and this enzyme can be used as a biological fungicide against phytopathogenic fungi, as well as an insecticide against insect pests.
Methods
In this study, 158 isolates, which were derived from bacteria cultures isolated from leaves and root rhizospheres of certain plants in Turkey, were selected after confirming that they are not phytopathogenic based on the hypersensitivity test performed on tobacco; and antifungal activity test was performed against Fusarium culmorum, which is a pathogenic fungi that cause decomposition of roots of vegetables. Accordingly, chitinase enzyme activity assay was performed on 31 isolates that have an antifungal activity, and among them the isolate of Bacillus subtilis TV-125 was selected, which has demonstrated the highest activity.
Results
Chitinase enzyme was purified by using ammonium sulphate and DEAE-sephadex ion exchange chromatography. Ammonium sulphate precipitation of chitinase enzyme from Bacillus subtilis TV-125 isolate was performed at maximum range of 0-20%, and 28.4-fold purification was obtained with a 13.4% of yield. Optimum activity of the purified enzyme was observed at pH 4.0 and at 50°C of temperature. In addition, it was identified that Bacillus subtilis TV-125A isolate retains 42% of its activity at 80°C temperature.
Conclusion
In the last phase of the study, chitinase enzyme purified from Bacillus subtilis TV-125A was tested on four fungal agents, although all the results were positive, it was particularly effective on F. culmorum according to the findings.
doi:10.1186/s12941-014-0035-3
PMCID: PMC4236515  PMID: 25112904
Chitinase; Purification; Antifungal activity; Fusarium culmorum; Bacillus subtilis
28.  Type II and type IV topoisomerase mutations in clinical isolates of Morganella morganii harbouring the qnrD gene 
Introduction
The aim of this study was to show the emergence of the qnrD gene among fluoroquinolone-resistant Morganella morganii isolate. The occurrence of mutations in DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC,parE) genes was also investigated in this strain.
Methodology
95 clinical Enterobacteria were screened for harbouring the qnrD gene. The clinical isolate of M. morganii was recovered from urine from a patient hospitalized in the urology unit at Fattouma Bourguiba Hospital, Tunisia. Antibiotic susceptibility was tested with the agar disk diffusion method. Quinolone susceptibility was studied with microbroth dilution technique. The investigations of plasmid mediated quinolone resistance (PMQR) and topoisomerases mutations were performed by polymerase chain reaction and nucleotide sequencing.
Results
This isolate showed high level of resistance to quinolones. The MIC with microbroth dilution technique was 512 μg/ml for norfloxacin, 256 μg/ml for ofloxacin and ciprofloxacin and 64μg/ml for levofloxacin.
This strain was found to harbour the quinolone resistance determinant qnrD. In addition, this strain harboured two new gyrB mutations (S463A, S464Y) and one parC mutation (S80I).
Conclusions
This is the first report in Tunisia of qnrD determinant and tow new gyrB muations in M. morganii. The nosocomial infection due to this proteeae invites further study of its epidemiologic evolution.
doi:10.1186/s12941-014-0034-4
PMCID: PMC4236555  PMID: 25106550
Morganella morganii; Quinolone resistance; Topoisomerase mutation; qnrD
29.  Polymerase chain reaction–based assays for the diagnosis of human brucellosis 
Polymerase chain reaction (PCR) is an in vitro technique for the nucleic acid amplification, which is commonly used to diagnose infectious diseases. The use of PCR for pathogens detection, genotyping and quantification has some advantages, such as high sensitivity, high specificity, reproducibility and technical ease. Brucellosis is a common zoonosis caused by Brucella spp., which still remains as a major health problem in many developing countries around the world. The direct culture and immunohistochemistry can be used for detecting infection with Brucella spp. However, PCR has the potential to address limitations of these methods. PCR are now one of the most useful assays for the diagnosis in human brucellosis. The aim of this review was to summarize the main PCR techniques and their applications for diagnosis and follow-up of patients with brucellosis. Moreover, advantages or limitation of the different PCR methods as well as the evaluation of PCR results for treatment and follow-up of human brucellosis were also discussed.
doi:10.1186/s12941-014-0031-7
PMCID: PMC4236518  PMID: 25082566
Polymerase chain reaction techniques; Human brucellosis; Brucella spp; Molecular diagnosis
30.  Novel approach of using a cocktail of designed bacteriophages against gut pathogenic E. coli for bacterial load biocontrol 
Background
This study was conducted to explore new approaches of animal biocontrol via biological control feed.
Method
White rats were subjected to 140 highly lytic designed phages specific against E. coli. Phages were fed via drinking water, oral injection, and vegetable capsules. Phage feeding was applied by 24 h feeding with 11d monitoring and 20d phage feeding and monitoring. Group of rats received external pathogenic E. coli and another group did not, namely groups A and B.
Results
Phage feeding for 20d via vegetable capsules yielded the highest reduction of fecal E. coli, 3.02 and 4.62 log, in rats group A and B respectively. Second best, feeding for 20d via drinking water with alkali yielded 2.78 and 4.08 log in rats groups A and B respectively. The peak reduction in E. coli output was 5–10 d after phage feeding. Phage control declined after 10th day of feeding.
Conclusions
The use of cocktail of designed phages succeeded in suppressing flora or external E. coli. The phage feed biocontrol is efficient in controlling E. coli at the pre-harvest period, precisely at the 6th-8th day of phage feeding when the lowest E. coli output found.
doi:10.1186/s12941-014-0039-z
PMCID: PMC4222638  PMID: 25062829
Bacteriophage; Animal feed; Biocontrol; E. coli; Bioprocessing; Phage design; Phage cocktail
31.  Evaluation of the anti-Listeria potentials of some plant-derived triterpenes 
Background
Listeriosis is a fatal disease caused by pathogenic Listeria bacteria and it is most prevalent in immune-compromised individuals. The increase in numbers of immune-compromised individuals against a background of Listeria antibiotic resistance, limits listeriosis treatment options. This therefore calls for research into substitute treatments, of which, medicinal plants derived compounds offer a viable alternative.
Methods
The broth microdilution assay was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of three plant triterpenes namely 3β-hydroxylanosta-9,24-dien-21-oic acid, methyl-3β-hydroxylanosta-9,24-dien-21-oate and 3β-acetylursolic acid, against Listeria monocytogenes, Listeria ivanovii and Listeria grayi species. The chequerboard method was used to assess the interactions between the triterpenes and conventional antibiotics: ampicillin, neomycin, gentamicin and penicillin G. The lactate dehydrogenase membrane damage method was used to assess the triterpenes’ membrane damaging potentials against the Listeria bacteria.
Results
The triterpenes’ MIC values were found to range from 0.185 to 1.67 mg/ml while, the MBC determination assay results revealed that the test triterpenes were bacteriostatic against the Listeria bacteria. The interactions involving 3β-hydroxylanosta-9,24-dien-21-oic acid were mainly additive with ampicillin and synergistic with neomycin, gentamicin and penicillin G. The interactions involving methyl-3β-hydroxylanosta-9,24-dien-21-oate were mainly antagonistic with ampicillin, indifferent with neomycin, ranging from synergistic to indifference with gentamicin and synergistic with penicillin G. The interactions involving 3β-acetylursolic acid were mainly indifferent with ampicillin, synergistic with neomycin and gentamicin while ranging between synergistic and additive with penicillin G. The low levels of cytosolic lactate dehydrogenase released from the cells treated with 4× MIC concentration of the triterpenes in comparison to that of cells treated with 3% Triton X-100 proved that membrane damage was not the mode of action of the triterpenes.
Conclusion
This study therefore shows the potential that these plant triterpenes have in listeriosis chemotherapy especially as shown by the favourable interactions they had with penicillin G, one of the antibiotics of choice in listeriosis treatment.
doi:10.1186/s12941-014-0037-1
PMCID: PMC4115164  PMID: 25056181
3β-hydroxylanosta-9,24-dien-21-oic acid; Methyl-3β-hydroxylanosta-9,24-dien-21-oate; 3β-acetylursolic acid; Listeria; Protorhus longifolia; Mimusops caffra
32.  Rapid genetic typing of diarrheagenic Escherichia coli using a two-tube modified molecular beacon based multiplex real-time PCR assay and its clinical application 
Background
Diarrheagenic Escherichia coli (DEC), including Enterotoxigenic E.coli (ETEC), Enteroaggregative E.coli (EAEC), Enteropathogenic E.coli (EPEC), Enterohemolysin E.coli (EHEC) and Enteroinvasive E.coli (EIEC) causes diarrhea or hemolytic uremic syndromes among infants and travelers around the world. A rapid, reliable and repeatable method is urgent for identifying DEC so as to provide the reference for responding to diarrheal disease outbreak and the treatment of the diarrheal patients associated with DEC.
Methods
In this study, specific primers and modified molecular beacon probes of nine specific virulence genes, whose 5′end were added with homo tail sequence, were designed; and a two-tube modified molecular beacon based multiplex real–time PCR (rtPCR) assay for the identification of five Escherichia coli pathotypes, including ETEC, EAEC, EPEC, EHEC and EIEC was developed and optimized. Totally 102 bacterial strains, including 52 reference bacterial strains and 50 clinical strains were detected to confirm whether the target genes selected were specific. Then detection limits of the assay were tested. Lastly, the assay was applied to the detection of 11860 clinical samples to evaluate the specificity and sensitivity of the developed assay compared with the conventional PCR.
Results
The target genes were 100% specific as assessed on 102 bacterial strains since no cross-reactions were observed. The detection limits ranged from 88 CFU/mL (EHEC) to 880 CFU/mL (EPEC). Compared with the conventional PCR, the specificity and sensitivity of the multiplex rtPCR was 100% and over 99%, respectively. The coefficient of variation (CV) for each target gene ranged from 0.45% to 1.53%. 171 positive clinical samples were mostly identified as ETEC (n = 111, 64.9%) and EPEC (n = 38, 22.2%), which were the dominating pathotypes of DEC strains.
Conclusion
The developed multiplex rtPCR assay for the identification of DEC was high sensitive and specific and could be applied to the rapid identification of DEC in clinical and public health laboratories.
doi:10.1186/s12941-014-0030-8
PMCID: PMC4115161  PMID: 25023669
Diarrheagenic Escherichia coli; Two-tube multiplex real-time PCR; Modified molecular beacon; Identification; Application
33.  In vitro antimicrobial activity of Medilox® super-oxidized water 
Aim
Super-oxidized water is one of the broad spectrum disinfectants, which was introduced recently. There are many researches to find reliable chemicals which are effective, inexpensive, easy to obtain and use, and effective for disinfection of microorganisms leading hospital infections. Antimicrobial activity of super-oxidized water is promising. The aim of this study was to investigate the in-vitro antimicrobial activity of different concentrations of Medilox® super-oxidized water that is approved by the Food and Drug Administration (FDA) as high level disinfectant.
Material and methods
In this study, super-oxidized water obtained from Medilox® [Soosan E & C, Korea] device, which had been already installed in our hospital, was used. Antimicrobial activities of different concentrations of super-oxidized water (1/1, 1/2, 1/5, 1/10, 1/20, 1/50, 1/100) at different exposure times (1, 2, 5, 10, 30 min) against six ATCC strains, eight antibiotic resistant bacteria, yeasts and molds were evaluated using qualitative suspension test. Dey-Engley Neutralizing Broth [Sigma-Aldrich, USA] was used as neutralizing agent.
Results
Medilox® was found to be effective against all standard strains (Acinetobacter baumannii 19606, Escherichia coli 25922, Enterococcus faecalis 29212, Klebsiella pneumoniae 254988, Pseudomonas aeruginosa 27853, Staphylococcus aureus 29213), all clinical isolates (Acinetobacter baumannii, Escherichia coli, vancomycin-resistant Enterococcus faecium, Klebsiella pneumoniae, Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, Bacillus subtilis, Myroides spp.), and all yeastsat 1/1 dilution in ≥ 1 minute. It was found to be effective on Aspergillus flavus at 1/1 dilution in ≥ 2 minutes and on certain molds in ≥ 5 minutes.
Conclusion
Medilox® super-oxidized water is a broad spectrum, on-site producible disinfectant, which is effective on bacteria and fungi and can be used for the control of nosocomial infection.
doi:10.1186/1476-0711-13-29
PMCID: PMC4107540  PMID: 25023905
Super-oxidized water; Medilox; Disinfectant; Bacteria; Fungi
34.  The effect of indomethacin, myeloperoxidase, and certain steroid hormones on bactericidal activity: an ex vivo and in vivo experimental study 
Background
The role of myeloperoxidase (MPO) is essential in the killing of phagocytosed bacteria. Certain steroid hormones increase MPO plasma concentration. Our aim was to test the effect of MPO, its inhibitor indomethacin, and certain steroid hormones on bactericidal activity.
Methods
Human polymorphonuclear leukocytes (PMN) were incubated with opsonised Escherichia coli and either MPO, indomethacin, estradiol, or hydrocortisone. Intracellular killing capacity was evaluated with UV microscopy after treatment with fluorescent dye. Next, an in vivo experiment was performed with nine groups of rats: in the first phase of the study indomethacin treatment and Pasteurella multocida infection (Ii), indomethacin treatment without infection (I0), untreated control with infection (Mi) and untreated control without infection (M0); in the second phase of the study rats with infection and testosterone treatment (NT), castration, infection and testosterone treatment (CT), castration, infection and estradiol treatment (CE), non-castrated infected control (N0), and castrated infected control (C0). After treatment bacteria were reisolated from the liver and heart blood on agar plates, and laboratory parameters were analyzed. For the comparison of laboratory results ANOVA or Kruskal-Wallis test and LSD post hoc test was used.
Results
Indomethacin did not have a remarkable effect on the bacterial killing of PMNs, while the other compounds increased bacterial killing to various degrees. In the animal model indomethacin and infection caused a poor clinical state, a great number of reisolated bacteria, elevated white blood cell (WBC) count, decreased C-reactive protein (CRP) and serum albumin levels. Testosterone treatment resulted in less bacterial colony numbers in group NT, but not in group CT compared to respective controls (N0, C0). Estradiol treatment (CE) decreased colony numbers compared to control (C0). Hormone administration resulted in lower WBC counts, and in group CE, a decreased CRP.
Conclusions
MPO, estradiol, and hydrocortisone improve bacterial killing activity of PMNs. Indomethacin treatment and castration weaken immune responses and clinical state of infected rats, while testosterone and estradiol have a beneficial effect.
doi:10.1186/1476-0711-13-27
PMCID: PMC4105879  PMID: 25001579
Bacterial killing activity; Infection; Myeloperoxidase; Indomethacin; Testosterone; Estradiol; Hydrocortisone
35.  Antifungal activity of synthetic naphthoquinones against dermatophytes and opportunistic fungi: preliminary mechanism-of-action tests 
This study evaluated the antifungal activities of synthetic naphthoquinones against opportunistic and dermatophytic fungi and their preliminary mechanisms of action. The minimum inhibitory concentrations (MICs) of four synthetic naphthoquinones for 89 microorganisms, including opportunistic yeast agents, dermatophytes and opportunistic filamentous fungi, were determined. The compound that exhibited the best activity was assessed for its action against the cell wall (sorbitol test), for interference associated with ergosterol interaction, for osmotic balance (K+ efflux) and for membrane leakage of substances that absorb at the wavelength of 260 nm. All tested naphthoquinones exhibited antifungal activity, and compound IVS320 (3a,10b-dihydro-1H-cyclopenta [b] naphtho [2,3-d] furan-5,10-dione)-dione) demonstrated the lowest MICs across the tested species. The MIC of IVS320 was particularly low for dermatophytes (values ranging from 5–28 μg/mL) and Cryptococcus spp. (3–5 μg/mL). In preliminary mechanism-of-action tests, IVS320 did not alter the fungal cell wall but did cause problems in terms of cell membrane permeability (efflux of K+ and leakage of substances that absorb at 260 nm). This last effect was unrelated to ergosterol interactions with the membrane.
doi:10.1186/1476-0711-13-26
PMCID: PMC4094904  PMID: 24998949
Naphthoquinones; Antifungal activity; Mechanism of action
36.  Nasopharyngeal carriage of Staphylococcus aureus among imprisoned males from Brazil without exposure to healthcare: risk factors and molecular characterization 
Background
Previous studies report high prevalence of Methicillin-resistant Staphylococcus aureus (MRSA) colonization among imprisoned populations. However, there are no data on that prevalence in Brazilian correctional institutions.
Findings
We tested 302 male prisoners for nasopharyngeal colonization with Staphylococcus aureus from February 2009 through April 2010. The overall isolation rate of S. aureus was 16.5% (50/302). Men who had sex with men, users of inhalatory drugs and those with previous lung or skin diseases were more likely to be colonized with S. aureus. MRSA was isolated from 0.7% of subjects (2/302). The two Community-associated (CA)-MRSA belonged to ST5 but were unrelated based on the PFGE results. Both harbored SCCmec IV, and did not possess the Panton-Valentine Leukocidin gene.
Conclusion
We found low prevalence of S. aureus and CA-MRSA among prisoners. MRSA isolates ST5 from two subjects harboured SCCmec IV and presented different PFGE patterns.
doi:10.1186/1476-0711-13-25
PMCID: PMC4099405  PMID: 24990470
Staphylococcus aureus; MRSA; Inmates
37.  3,4-DHPEA-EA from Olea Europaea L. is effective against standard and clinical isolates of Staphylococcus sp. 
Background
The aim of the present work was to evaluate the antibacterial effect of 3,4-DHPEA-EA (methyl-4-(2-(3,4-dihydroxyphenethoxy)-2-oxoethyl)-3-formyl-2-methyl-3,4-dihydro-2H-pyran-5-carboxylate), a derivate of oleuropein, against a range of Gram-positive bacteria, including ATCC strains, food and clinical isolates.
Methods
The minimum inhibitory concentrations (MICs) of 3,4-DHPEA-EA were determined by the broth microdilution method and the Bioscreen C.
Results
3,4-DHPEA-EA was effective against ATCC and clinical isolates of Staphylococcus aureus (MIC values between 125 and 250 μg/ml) and ATCC and clinical isolates of Staphylococcus epidermidis (MIC values between 7.81 and 62.5 μg/ml). No significant differences were observed between the two solvents (methanol and DMSO) used to dissolve 3,4-DHPEA-EA.
Conclusions
The results obtained could be used to develop novel therapies for the treatment of skin infections. Further studies need to be performed to elucidate the formation of 3,4-DHPEA-EA by acid hydrolysis of oleuropein in the human stomach.
doi:10.1186/1476-0711-13-24
PMCID: PMC4107751  PMID: 24986240
Olea europaea L; 3,4-DHPEA-EA; Antimicrobial; Staphylococci
38.  Acute liver failure caused by severe acute hepatitis B: a case series from a multi-center investigation 
Background
Few data can be available regarding acute liver failure (ALF) caused by severe acute hepatitis B up to now. This study aims to report such cases from China.
Findings
We conducted a multi-center investigation on ALF from 7 tertiary hospitals in different areas of China. A total of 11 patients with ALF caused by severe acute hepatitis B were finally identified. In these patients, there were 10 male and 1 female patients. As a serious complication, apparent hemorrhage occurred in 9 patients. Eventually, in these 11 patients, 4 survived and 7 died. 4 died of heavy bleeding, 2 died of systemic inflammatory response syndrome and 1 died of irreversible coma. No patients received liver transplantation.
Conclusions
ALF caused by severe acute hepatitis B is worthy of formal studies based on its rarity and severity.
doi:10.1186/1476-0711-13-23
PMCID: PMC4077644  PMID: 24958233
Acute liver failure; Acute hepatitis B; Prognosis
39.  Fecal carriage of extended-spectrum β-lactamases and AmpC-producing Escherichia coli in a Libyan community 
Background
Extended-spectrum β-lactamases (ESBLs), including the AmpC type, are important mechanisms of resistance among Enterobacteriaeceae. CTX-M type extended-spectrum β- lactamases, of which there are now over 90 variants, are distributed globally, yet appear to vary in regional distribution. AmpC β-lactamases hydrolyze third generation cephalosporins, but are resistant to inhibition by clavulanate or other β-lactamase inhibitors in vitro. Fecal carriage and rates of colonization by bacteria harboring these resistance mechanisms have been reported in patients with community-acquired infections and in healthy members of their households. Expression of these ESBLs compromises the efficacy of current antibacterial therapies, potentially increasing the seriousness of hospital- and community-acquired Escherichia coli (E. coli) infections.
To investigate the occurrence of ESBL-producing E. coli in human fecal flora isolated from two pediatric populations residing in the Libyan cities Zleiten and Abou El Khoms. Isolates were further studied to characterize genes encoding β-lactam resistance, and establish genetic relationships.
Methods
Antibiotic resistance profiles of phenotypically characterized E. coli isolates recovered from the stools of 243 Libyan children during two surveillance periods in 2001 and 2007 were determined by the disk diffusion method. ESBL-screening was performed using the cephalosporin/clavulanate double synergy disc method, and the AmpC-phenotype was confirmed by the aminophenyl-boronic acid test. ESBL genes were molecularly characterized. Phylogenetic group and multilocus sequence typing (MLST) were determined for ESBL-producing isolates and PFGE was performed to compare banding profiles of some dominant strains.
Results
ESBLs were identified in 13.4% (18/134) of E. coli isolates, and nine isolates (6.7%) demonstrated AmpC activity; all 18 isolates contained a CTX-M gene. Three CTX-M gene families (CTX-M-1, n = 9; CTX-M-15, n = 8 and CTX-M-3, n = 1) were distributed in diverse E. coli backgrounds (phylogenetic group D, 39%; B2, 28%; B1, 22% and A, 11%). MLST analysis revealed 14 sequence type (ST) with six new sequence types. The gene encoding the CMY-2 enzyme was detected in five AmpC-positive E. coli.
Conclusions
These results identified heterogeneous clones of CTX-M-producing E. coli in the fecal isolates, indicating that the intestinal tract acts as a reservoir for ESBL-producing organisms, and a trafficker of antibiotic resistance genes.
doi:10.1186/1476-0711-13-22
PMCID: PMC4107601  PMID: 24934873
ESBLs; Libya; CTXM; AmpC; Fecal carriage; E. coli; MLST
40.  Study of the frequency of Clostridium difficile tcdA, tcdB, cdtA and cdtB genes in feces of Calves in south west of Iran 
Background
Clostridium difficile (C. difficile) is a gram-positive, toxin-producing bacillus which is an intestinal pathogen in both humans and animals and causes a range of digestive disorders including inflammation of the bowel, abdominal pain, fever and diarrhea. C. difficile toxins include enterotoxin (Toxin A), cytotoxin (Toxin B) and a binary toxin. Two large protein toxins A and B are encoded by separate genes, tcdA and tcdB. Clostridium difficile infection (CDI) mainly caused by the activity of the genes tcdA and tcdB. The binary toxin is encoded by the genes cdtA and cdtB. The binary toxin caused increased adherence of bacteria to intestinal epithelium. The aim of the present study was isolation of C. difficile from feces of calves, and study of the frequency of C. difficile virulence genes.
Methods
150 samples of fresh feces from calves were collected and C. difficile was isolated from feces of calves using bacterial culture methods. DNA was extracted by a genomic DNA purification kit. Then PCR method was used for definitive diagnosis of C. difficile. Multiplex PCR method performed for identification of tcdA, tcdB, cdtA and cdtB genes. In the final stage antimicrobial resistance determining was carried out by standard Bauer-Kirby disk diffusion method.
Results
C. difficile was isolated from 90 samples (60%). The tcdA was observed in 8 isolates (8.8%), tcdB in 16 isolates (17.7%), cdtA in 8 isolates (8.8%) and cdtB in 14 isolates (15.5%). Only 1 isolated (1.1%) was containing all four genes tcdA, tcdB, cdtA and cdtB, 2 isolates (2.2%) only had both tcdA and tcdB genes, and there was no sample positive only for both cdtA and cdtB. The highest rate of drug resistance was against clindamycin (100%) and the highest rate of drug sensitivity was against ciprofloxacin (50%).
Conclusion
The results showed high incidence of C. difficile and also high antibiotic resistance of this bacterium, but frequency of strains containing virulence genes (tcdA, tcdB, cdtA and cdtB) was low.
doi:10.1186/1476-0711-13-21
PMCID: PMC4060091  PMID: 24903619
Clostridium difficile; Calves; Toxin A; Toxin B; Binary toxin
41.  Genetic characteristics and antimicrobial resistance of Staphylococcus epidermidis isolates from patients with catheter-related bloodstream infections and from colonized healthcare workers in a Belgian hospital 
Background
Staphylococcus epidermidis is a pathogen that is frequently encountered in the hospital environment. Healthcare workers (HCWs) can serve as a reservoir for the transmission of S. epidermidis to patients.
Methods
The aim of this study was to compare and identify differences between S. epidermidis isolated from 20 patients with catheter-related bloodstream infections (CRBSIs) and from the hands of 42 HCWs in the same hospital in terms of antimicrobial resistance, biofilm production, presence of the intercellular adhesion (ica) operon and genetic diversity (pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and staphylococcal cassette chromosome (SCC) mec typing).
Results
S. epidermidis isolates that caused CRBSI were resistant to significantly more non-betalactam drugs than were isolates collected from HCWs. Among the 43 mecA positive isolates (26 from HCWs), the most frequent SCCmec type was type IV (44%). The ica operon was significantly more prevalent in CRBSI isolates than in HCWs (P < 0.05). Weak in vitro biofilm production seemed to correlate with the absence of the ica operon regardless of the commensal or pathogenic origin of the isolate. The 62 isolates showed high diversity in their PFGE patterns divided into 37 different types: 19 harbored only by the CRBSI isolates and 6 shared by the clinical and HCW isolates. MLST revealed a total of ten different sequence types (ST). ST2 was limited to CRBSI-specific PFGE types while the “mixed” PFGE types were ST5, ST16, ST88 and ST153.
Conclusion
One third of CRBSI episodes were due to isolates belonging to PFGE types that were also found on the hands of HCWs, suggesting that HCW serve as a reservoir for oxacillin resistance and transmission to patients. However, S. epidermidis ST2, mecA-positive and icaA-positive isolates, which caused the majority of clinically severe CRBSI, were not recovered from the HCW’s hands.
doi:10.1186/1476-0711-13-20
PMCID: PMC4066695  PMID: 24899534
Staphylococcus epidermidis; Catheter-related bloodstream infection; Molecular epidemiology; PFGE; MLST
42.  Manuka honey treatment of biofilms of Pseudomonas aeruginosa results in the emergence of isolates with increased honey resistance 
Background
Medical grade manuka honeys are well known to be efficacious against Pseudomonas aeruginosa being bactericidal and inhibiting the development of biofilms; moreover manuka honey effectively kills P. aeruginosa embedded within an established biofilm. Sustained honey resistance has not been previously documented for planktonic or biofilm P. aeruginosa.
Methods
Minimum inhibitory concentrations for manuka honey and antibiotics were determined using broth micro-dilution methods. Minimum biofilm eliminating concentrations (MBEC) and biofilm biomass were determined using the crystal violet method. Sub-culture used non-selective media and the grid-plate method.
Results
When honey treated biofilm biomass of two strains of P. aeruginosa (reference strain ATCC 9027 and the clinical isolate 867) were sub-cultured onto non-selective media isolates emerged that exhibited reduced susceptibility to manuka honey. Significantly, this characteristic was sustained with repeated sub-culture onto non-selective media resulting in increased minimum inhibitory concentrations (MIC) of between 5-7% (w/v) and increased minimum biofilm eliminating concentrations (MBEC) of up to 15% (w/v). Interestingly the resistant isolates showed reduced susceptibility to antibiotic treatment with rifampicin and imipenem as well as being more prolific biofilm-formers than the progenitor strains.
Conclusions
P. aeruginosa biofilms treated with manuka honey equivalent to the MBEC harbour slow growing, viable persistor organisms that exhibit sustained, increased resistance to manuka honey and antibiotic treatment, suggesting a shared mechanism of resistance. This sheds new light on the propensity for biofilm embedded organisms to resist honey treatment and become persistor organisms that are tolerant to other antimicrobial therapies.
doi:10.1186/1476-0711-13-19
PMCID: PMC4023514  PMID: 24884949
Antimicrobial; Small colony variant
43.  The polyene antifungals, amphotericin B and nystatin, cause cell death in Saccharomyces cerevisiae by a distinct mechanism to amphibian-derived antimicrobial peptides 
Background
There is a pressing need to identify novel antifungal drug targets to aid in the therapy of life-threatening mycoses and overcome increasing drug resistance. Identifying specific mechanisms of action of membrane-interacting antimicrobial drugs on the model fungus Saccharomyces cerevisiae is one avenue towards addressing this issue. The S. cerevisiae deletion mutants Δizh2, Δizh3, Δaif1 and Δstm1 were demonstrated to be resistant to amphibian-derived antimicrobial peptides (AMPs). The purpose of this study was to examine whether AMPs and polyene antifungals have a similar mode of action; this was done by comparing the relative tolerance of the mutants listed above to both classes of antifungal.
Findings
In support of previous findings on solid media it was shown that Δizh2 and Δizh3 mutants had increased resistance to both amphotericin B (1–2 μg ml−1) and nystatin (2.5 – 5 μg ml−1) in liquid culture, after acute exposure. However, Δaif1 and Δstm1 had wild-type levels of susceptibility to these polyenes. The generation of reactive oxygen species (ROS) after exposure to amphotericin B was also reduced in Δizh2 and Δizh3. These data indicated that polyene antifungal and AMPs may act via distinct mechanisms of inducing cell death in S. cerevisiae.
Conclusions
Further understanding of the mechanism(s) involved in causing cell death and the roles of IZH2 and IZH3 in drug susceptibility may help to inform improved drug design and treatment of fungal pathogens.
doi:10.1186/1476-0711-13-18
PMCID: PMC4036090  PMID: 24884795
Amphotericin B; Antifungal resistance; YOL002C; Antifungals
44.  Recurrent paratyphoid fever A co-infected with hepatitis A reactivated chronic hepatitis B 
We report here a case of recurrent paratyphoid fever A with hepatitis A co-infection in a patient with chronic hepatitis B. A 26-year-old male patient, who was a hepatitis B virus carrier, was co-infected with Salmonella enterica serovar Paratyphi A and hepatitis A virus. The recurrence of the paratyphoid fever may be ascribed to the coexistence of hepatitis B, a course of ceftriaxone plus levofloxacin that was too short and the insensitivity of paratyphoid fever A to levofloxacin. We find that an adequate course and dose of ceftriaxone is a better strategy for treating paratyphoid fever. Furthermore, the co-infection of paratyphoid fever with hepatitis A may stimulate cellular immunity and break immunotolerance. Thus, the administration of the anti-viral agent entecavir may greatly improve the prognosis of this patient with chronic hepatitis B, and the episodes of paratyphoid fever and hepatitis A infection prompt the use of timely antiviral therapy.
doi:10.1186/1476-0711-13-17
PMCID: PMC4055195  PMID: 24884719
Recurrent paratyphoid fever A; Hepatitis A; Chronic hepatitis B reactivation
45.  Molecular characterization of clinical multidrug-resistant Klebsiella pneumoniae isolates 
Background
Klebsiella pneumoniae is a frequent nosocomial pathogen, with the multidrug-resistant (MDR) K. pneumoniae being a major public health concern, frequently causing difficult-to-treat infections worldwide. The aim of this study was to investigate the molecular characterization of clinical MDR Klebsiella pneumoniae isolates.
Methods
A total of 27 non-duplicate MDR K. pneumoniae isolates with a CTX-CIP-AK resistance pattern were investigated for the prevalence of antimicrobial resistance genes including extended spectrum β-lactamase genes (ESBLs), plasmid-mediated quinolone resistance (PMQR) genes, 16S rRNA methylase (16S-RMTase) genes, and integrons by polymerase chain reaction (PCR) amplification and DNA sequencing. Plasmid replicons were typed by PCR-based replicon typing (PBRT). Multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were carried out to characterize the strain relatedness.
Results
All the isolates co-harbored 3 or more resistance determinants. OqxAB, CTX-M-type ESBLs and RmtB were the most frequent determinants, distributed among19 (70.4%),18 (66.7%) and 8 (29.6%) strains. Fourteen isolates harbored class 1 integrons, with orfD-aacA4 being the most frequent gene cassette array. Class 3 integrons were less frequently identified and contained the gene cassette array of blaGES-1-blaOXA-10-aac(6′)-Ib. IncFII replicon was most commonly found in this collection. One cluster was observed with ≥80% similarity among profiles obtained by PFGE, and one sequence type (ST) by MLST, namely ST11, was observed in the cluster.
Conclusion
K. pneumoniae carbapenemase (KPC)–producing ST11 was the main clone detected. Of particular concern was the high prevalence of multiple resistance determinants, classs I integrons and IncFII plasmid replicon among these MDR strains, which provide advantages for the rapid development of MDR strains.
doi:10.1186/1476-0711-13-16
PMCID: PMC4030571  PMID: 24884610
Multidrug resistance; Resistance determinants; Multi-locus sequence typing; Pulsed-field gel electrophoresis; Plasmid replicons
46.  Prescription antibiotics for outpatients in Bangladesh: a cross-sectional health survey conducted in three cities 
Background
Antibiotics prescribing by physicians have gained due importance across the globe, mainly because of an increase in antibiotic usage, prevalence of infections and drug resistances. The present study is aimed to evaluate the physicians prescribing pattern of antibiotics, their usages by outpatients and disease conditions for which the antibiotics are prescribed in three cities of Bangladesh.
Methods
This cross sectional health survey was carried out with a self designed standard questionnaire by manual data collection over a three months period (20.03.2013 to 20.06.2013) at three adjacent cities Jessore Sadar, Monirampur and Keshabpur upazila respectively. The data were collected from the patient’s prescription and by directly interviewing the patients who were prescribed at least one antibiotic during the study period. WHO Anatomical Therapeutic Chemical (ATC) classifications for antibiotics was used and descriptive statistics were applied to the collected data and analyzed using Microsoft Excel software. Modified Wald method was applied to calculate 95% CI.
Results
A total of 900 prescriptions were analyzed during the study period. It was found that the prescriber prescribed antibiotics to the patients who were suffering mainly from cold and fever, infections, diarrhea and gonorrhea. The highest prescribed antibiotic groups were cephalosporins (31.78%), macrolides (27.33%), quinolones (16.33%), penicillins (7.11%), and metronidazoles (6.78%) respectively. Two or more antibiotics were prescribed in 25.44% of prescriptions. A total of 66.89% prescriptions had complete information on dosage form, 57% had complete direction for antibiotics use and 64.22% patients completed full course of antibiotics. Although 83% prescriptions have no clinical test for using antibiotics, even though the percentages of patients’ disease recovery were 61.78% and incompliance were 38.22%.
Conclusion
From this research, it is observed that physicians prescribed antibiotics rationally in some cases but needs to ensure in all cases of prescription. Because irrational use leads to the spread of bacterial resistance to antibiotics and related health problems, our findings have important implications for public education and the enforcement of regulations regarding the prescription of antibiotics in Bangladesh.
doi:10.1186/1476-0711-13-15
PMCID: PMC4004450  PMID: 24755269
Antibiotics; Prescription patterns; Antibiotic resistance; Bangladesh
47.  Antimicrobial susceptibility pattern of bacterial isolates from wound infection and their sensitivity to alternative topical agents at Jimma University Specialized Hospital, South-West Ethiopia 
Background
Wound infection is one of the health problems that are caused and aggravated by the invasion of pathogenic organisms. Information on local pathogens and sensitivity to antimicrobial agents, and topical agents like acetic acid is crucial for successful treatment of wounds.
Objectives
To determine antimicrobial susceptibility pattern of bacterial isolates from wound infection and their sensitivity to alternative topical agents at Jimma University Specialized Hospital.
Methods
A cross sectional study was conducted among patients with wound infection visiting Jimma University Specialized Hospital, from May to September 2013. Wound swab was collected using sterile cotton swabs and processed for bacterial isolation and susceptibility testing to antimicrobial agents, acetic acid, hydrogen peroxide and dabkin solution following standard bacteriological techniques. Biochemical tests were done to identify the species of the organisms. Sensitivity testing was done using Kirby- Baur disk diffusion method. Minimum inhibitory and bactericidal concentration was done using tube dilution method.
Results
In this study 145 bacterial isolates were recovered from 150 specimens showing an isolation rate of 87.3%. The predominant bacteria isolated from the infected wounds were Staphylococcus aureus 47 (32.4%) followed by Escherichia coli 29 (20%), Proteus species 23 (16%), Coagulase negative Staphylococci 21 (14.5%), Klebsiella pneumoniae 14 (10%) and Pseudomonas aeruginosa 11 (8%). All isolates showed high frequency of resistance to ampicillin, penicillin, cephalothin and tetracycline. The overall multiple drug resistance patterns were found to be 85%. Acetic acid (0.5%), Dabkin solution (1%) and 3% hydrogen peroxide were bactericidal to all isolated bacteria and lethal effect observed when applied for 10 minutes.
Conclusions
On in vitro sensitivity testing, ampicillin, penicillin, cephalothin and tetracycline were the least effective. Gentamicin, norfloxacin, ciprofloxacin, vancomycin and amikacin were the most effective antibiotics. Acetic acid (0.5%), dabkin solution (1%) and H2O2 (3%) were bactericidal to all isolates.
doi:10.1186/1476-0711-13-14
PMCID: PMC4017222  PMID: 24731394
Bacterial pathogens; Drug resistance; Wound infection; Jimma; Ethiopia
48.  Intracellular activity of tedizolid phosphate and ACH-702 versus Mycobacterium tuberculosis infected macrophages 
Background
Due to the emergency of multidrug-resistant strains of Mycobacterium tuberculosis, is necessary the evaluation of new compounds.
Findings
Tedizolid, a novel oxazolidinone, and ACH-702, a new isothiazoloquinolone, were tested against M. tuberculosis infected THP-1 macrophages. These two compounds significantly decreased the number of intracellular mycobacteria at 0.25X, 1X, 4X and 16X the MIC value. The drugs were tested either in nanoparticules or in free solution.
Conclusion
Tedizolid and ACH-702 have a good intracellular killing activity comparable to that of rifampin or moxifloxacin.
doi:10.1186/1476-0711-13-13
PMCID: PMC3986449  PMID: 24708819
ACH-702; Tuberculosis; Oxazolidinones
49.  Asymmetric dimethylarginine levels in patients with cutaneous anthrax: a laboratory analysis 
Background
Asymmetric dimethylarginine (ADMA), the main endogenous inhibitor of nitric oxide synthase, is considered to be associated with endothelial dysfunction. High ADMA levels have been shown to be related with disorders causing vascular inflammation such as hypertension, hypercholesterolemia, atherosclerosis, chronic heart failure, stroke and sepsis. Cutaneous anthrax (CA) is a serious infectious disease which may cause vasculitis. The aim of the study was to investigate the serum ADMA levels in patients with CA.
Methods
A total of 35 serum samples of the patients with CA and 18 control sera were tested for ADMA levels using ADMA ELISA kit (Immunodiagnostik AG, Bensheim, Germany).
Results
ADMA levels were found to be significantly higher in the patients group than the controls (p < 0.001). In addition, ADMA levels were found to be positively associated with sedimentation rates (R = 0.413; p = 0.026), and inversely associated with international normalized ratio (INR) levels (R = -0.46; p = 0.011). A cut-off value of 0.475 of ADMA had a sensitivity of 74.3%, specificity of 77.8%, and accuracy of 75.5% in the diagnosis of CA.
Conclusion
Although the exact mechanism still remains unclear, ADMA levels could be related to immune activation in CA. In addition, these data might suggest the higher ADMA levels in patients could be due to the perivascular inflammation and vasculitis in CA.
doi:10.1186/1476-0711-13-12
PMCID: PMC3986940  PMID: 24669818
Dimethylarginine; Infection; Anthrax; Vasculitis
50.  Factors predicting prolonged empirical antifungal treatment in critically ill patients 
Objective
To determine the incidence, risk factors, and impact on outcome of prolonged empirical antifungal treatment in ICU patients.
Methods
Retrospective observational study performed during a one-year period. Patients who stayed in the ICU >48 h, and received empirical antifungal treatment were included. Patients with confirmed invasive fungal disease were excluded. Prolonged antifungal treatment was defined as percentage of days in the ICU with antifungals > median percentage in the whole cohort of patients.
Results
Among the 560 patients hospitalized for >48 h, 153 (27%) patients received empirical antifungal treatment and were included in this study. Fluconazole was the most frequently used antifungal (46% of study patients). Median length of ICU stay was 19 days (IQR 8, 34), median duration of antifungal treatment was 8 days (IQR 3, 16), and median percentage of days in the ICU with antifungals was 48% (IQR 25, 80). Seventy-seven patients (50%) received prolonged empirical antifungal treatment. Chemotherapy (OR [95% CI] 2.6 [1.07-6.69], p = 0.034), and suspected infection at ICU admission (3.1 [1.05-9.48], p = 0.041) were independently associated with prolonged empirical antifungal treatment.
Duration of mechanical ventilation and ICU stay were significantly shorter in patients with prolonged empirical antifungal treatment compared with those with no prolonged empirical antifungal treatment. However, ICU mortality was similar in the two groups (46 versus 52%, p = 0.62).
Conclusion
Empirical antifungal treatment was prescribed in a large proportion of study patients. Chemotherapy, and suspicion of infection at ICU admission are independently associated with prolonged empirical antifungal treatment.
doi:10.1186/1476-0711-13-11
PMCID: PMC3984712  PMID: 24621182
Antifungal treatment; Empirical treatment; Fungal infection; Invasive fungal disease; De-escalation

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