Isolation and antimicrobial susceptibility pattern of Staphylococcus aureus in patients with surgical site infection at Debre Markos Referral Hospital,Amhara Region,Ethiopia

Background

Staphylococcus aureus, especially Methicillin Resistant Staphylococcus Aureus (MRSA) is a major health problem recognized as the most important nosocomial pathogen, often causing postoperative wound infections. Antibiotic resistance by MRSA has grown to be common, and resistance to almost all antibiotics has been found among these strains. The aim of this study was to determine the prevalence, antimicrobial susceptibility patterns and associated risk factors of S. aureus in patients with surgical site infections in an Ethiopian hospital.

Methods

A cross-sectional study was conducted from December 1, 2011 to March 30, 2012 among patients with surgical site infections at Debre Markos Referral Hospital, Debre Markos, Ethiopia. All wound swabs obtained from patients with surgical site infections during the study period were cultured on mannitol salt agar media which is selective for S. aureus. Isolated strains of S. aureus were tested for antibiotic susceptibility patterns using standard disc diffusion technique, and interpretation of resistance was done based on Clinical and Laboratory Standard Institute criteria. Univariate and multivariable analyses were used to assess the risk factors.

Results

Of the 184 surgical patients who had developed surgical site infection, S. aureus was isolated from 73 (39.7%) cases. Out of the 73 isolates of S. aureus, 36 (49.7%) were MRSA. Among the study participants, prevalence of MRSA was found to be 19.6%. The clinical isolates showed >80% level of resistance to ampicillin, amoxicillin, penicillin G, erythromycin, gentamicin and cotrimoxazole whereas <50% level of resistance was observed against clindamycin, oxacillin, tetracycline and vancomycin. MRSA strains showed resistance ranging from 5.6% (vancomycin) to 100% (cotrimoxazole). Of the following risk factors: sex, age, pus consistency, duration of operation, type of surgery, ward and hospital stay, laparotomy type of surgery was identified as a risk factor for infection by S. aureus.

Conclusion

The prevalence of S. aureus and/or MRSA infection in surgical and gynaecology & obstetrics wards of Debre Markos Referral Hospital was found to be high. The majority of isolates were highly resistant to major antimicrobial agents.     

Prevalence of Antibiotic Resistance in Escherichia coli Fecal Isolates From Healthy Persons and Patients With Diarrhea

Objectives

This study aimed to investigate the prevalence of antibiotic resistance in fecal Escherichia coli isolates from healthy persons and patients with diarrhea.

Methods

E. coli isolates (n = 428) were obtained from fecal samples of apparently healthy volunteers and hospitalized patients with diarrhea. Susceptibility patterns of isolates to 16 antimicrobial agents were determined by agar disc diffusion.

Results

Most E. coli isolates exhibited less than 10% resistance against imipenem, cefotetan, aztreonam, cefepime, cefoxitin, amikacin and netilamicin, although greater than 65% were resistant to ampicillin and tetracycline. No significant difference in resistance rates for all tested antibiotics was found between isolates from the healthy-and diarrheal-patient groups, including for multi-drug resistance (p = 0.22). The highest number of resistant antibiotics was 12 antibiotics. No significant differences in antibiotic resistance were found among the sex and age strata for isolates from healthy individuals. However, antibiotic resistance rates to cefoxitin, cefotaxime, amikacin, and netilamicin were significantly higher in the isolates of men than those of women (p < 0.05) in isolates from patients with diarrhea. Furthermore, isolates from patients with diarrhea older than 40-years of age showed higher resistance to cefepime and aztreonam (p < 0.05).

Conclusion

High resistance to the antibiotics most frequently prescribed for diarrhea was found in isolates from patients with diarrhea and apparently healthy individuals without any significant difference.     

Molecular analysis and the toxin,MSCRAMM, and biofilm genes of methicillin-resistant Staphylococcus aureus strains isolated from pemphigus wounds: A study based on SCCmec and dru typing

IntroductionPemphigus is a chronic autoimmune blistering disease. Pemphigus blisters can damage the natural skin barrier and increase the risk of life-threatening conditions. Colonization of pemphigus wounds with methicillin-resistant Staphylococcus aureus (MRSA) prolongs wound healing and increases mortality rate. Assessing MRSA prevalence, types, and toxin and adhesion genes can facilitate the detection of MRSA strains which cause infections, selection of appropriate treatments, and healing of pemphigus wounds. This study aimed to determine the SCCmec, the direct repeat unit (dru) types (dts), and the toxin, MSCRAMM, and biofilm genes of MRSA strains isolated from pemphigus wounds.MethodsIn this cross-sectional study, 118 S. aureus isolates were gathered from 118 patients with pemphigus. MRSA detection was performed using the mecA gene. Using the polymerase chain reaction method, all MRSA isolates were assessed for the presence of the sea, seb, sec, tst, eta, pvl, hla, hlb, MSCRAMM, and ica genes. Typing and subtyping were performed through respectively SCCmec typing and dru typing methods. The Bionumerics software was used for analyzing the data and drawing the minimum spanning tree.FindingsFrom 118 S. aureus isolates, 51 were MRSA. SCCmec typing revealed the prevalence of SCCmec II with a prevalence of 64.7% (33 out of 51 isolates) and SCCmec III with a prevalence of 35.3% (18 out of 51 isolates). Dru typing indicated seven dts, namely dts 10a, 10g, 10m, 13i, 8h, 8i, and 9ca in two main clusters. The dt9ca was a new dru type and was registered in the dru-typing database (www.dru-typing.org). The prevalence rates of the hla, sea, and sec genes in MRSA isolates were respectively 54.9%, 27.4%, and 1.9%, while the hlb, seb, eta, and pvl genes were not detected at all. Only one MRSA with SCCmec III and dt10a carried the tst encoding gene. MSCRAMM gene analysis revealed the high prevalence of the eno (31.3%) and the fib (21.5%) genes. The prevalence rates of the icaA and icaD biofilm formation genes were 3.9% and 5.8%, respectively. There were no significant differences between the two detected SCCmec types and between the two detected dts clusters respecting the prevalence of the encoding genes of virulence factors and MSCRAMMs.ConclusionThe toxin genes hla and sea are prevalent among MRSA strains with SCCmec II and III isolated from pemphigus wounds. The most prevalent dts are dt10a and dt10g among MRSA with SCCmec III and dt8h and dt8i among MRSA with SCCmec II.     

A diversity of Antibiotic-resistant Staphylococcus spp. in a Public Transportation System

Objectives

Our goal was to determine the diversity and abundance of Staphylococcus bacteria on different components of a public transportation system in a mid-sized US city (Portland, Oregon) and to examine the level of drug resistance in these bacteria.

Methods

We collected 70 samples from 2 cm × 4 cm sections from seven different areas on buses and trains in Portland, USA, taking 10 samples from each area. We isolated a subset of 14 suspected Staphylococcus spp. colonies based on phenotype, and constructed a phylogeny from16S rRNA sequences to assist in identification. We used the Kirbye–Bauer disk diffusion method to determine resistance levels to six common antibiotics.

Results

We found a range of pathogenic Staphylococcus species. The mean bacterial colony counts were 97.1 on bus and train floors, 80.1 in cloth seats, 9.5 on handrails, 8.6 on seats and armrests at bus stops, 3.8 on the underside of seats, 2.2 on windows, and 1.8 on vinyl seats per 8 cm² sample area. These differences were significant (p < 0.001). Of the 14 isolates sequenced, 11 were staphylococci, and of these, five were resistant to penicillin and ampicillin, while only two displayed intermediate resistance to bacitracin. All 11 isolates were sensitive to trimethoprim-sulfamethoxazole, vancomycin, and tetracycline.

Conclusions

We found six different strains of Staphylococcus, and while there were varying levels of drug resistance, we did not find extensive levels of multidrug-resistant bacteria, and no S. aureus was found. We found floors and cloth seats to be areas on buses and trains that showed particularly high levels of bacteria.     

Expression of Recombinant pET22b-LysK-Cysteine/Histidine-Dependent Amidohydrolase/Peptidase Bacteriophage Therapeutic Protein in Escherichia coli BL21 (DE3)

Objectives

Bacteriophage-encoded endolysins are a group of enzymes that act by digesting the peptidoglycan of bacterial cell walls. LysK has been reported to lyse live staphylococcal cultures. LysK proteins containing only the cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) domain has the capability to show lytic activity against live clinical staphylococcal isolates, including methicillin-resistant Staphylococcus aureus (MRSA). The aim of this study was to clone and express LysK-CHAP domain in Escherichia coli BL21 (DE3) using pET22b as a secretion vector. The pET22b plasmid was used, which encoded a pelB secretion signal under the control of the strong bacteriophage T7 promoter.

Methods

The E. coli cloning strains DH5α and BL21 (DE3) were grown at 37°C with aeration in the Luria-Bertani medium. A plasmid encoding LysK-CHAP in a pET22b backbone was constructed. The pET22b vector containing LysK-CHAP sequences were digested with NcoI and HindIII restriction enzymes. Cloning accuracy was confirmed by electrophoresis. The pET22b-LysK plasmid was used to transform the E. coli strain BL21. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1mM to induce T7 RNA polymerase-based expression. Finally, western blot confirmed the expression of target protein.

Results

In this study, after double digestion of pEX and pET22b vectors with HindIII and NcoI, LysK gene was cloned into two HindIII and NcoI sites in pET22b vector, and then transformed to E. coli DH5α. Cloning was confirmed with double digestion and analyzed with agarose gel. The recombinant pET22b-LysK plasmid was transformed to E. coli BL21 and the expression was induced by IPTG. The expression was confirmed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting method. Observation of a 28.5 kDa band confirmed LysK protein expression.

Conclusion

In the present study, LysK-CHAP domain was successfully cloned and expressed at the pET22b vector and E. coli BL21 (DE3).     

Possibility of CTX-M-14 Gene Transfer from Shigella sonnei to a Commensal Escherichia coli Strain of the Gastroenteritis Microbiome

Objectives

To investigated whether the CTX-M-14 gene could be transferred from a clinical Shigella sonnei strain to commensal Escherichia coli strain in the gastroenteritis microbiome.

Methods

E. coli strains were isolated from 30 stool samples of S. sonnei infected students in a gastroenteritis outbreak in 2004 and were characterized by antibiotic resistance analysis, in vitro conjugation and in vivo transfer of CTX-M-14 gene and molecular assays.

Results

One strain of Escherichia coli that had high levels of resistance to cefotaxime was isolated from a patient infected with S. sonnei. Isoelectric focusing showed that the E. coli and S. sonnei strains produced a β-lactamase with an isoelectric point of 8.1. Moreover, polymerase chain reaction analysis indicated that both strains possessed the same DNA sequences for CTX-M-14. The results of in vitro and in vivo conjugation showed that the efficiency of CTX-M-14 transfer from S. sonnei to E. coli was similar to CTX-M-14 transfer between E. coli strains.

Conclusion

The data suggest that the acquisition of the extended-spectrum β-lactamases gene by pathogenic bacteria in the human intestinal tract to commensal microbiome bacteria can cause serious infectious diseases.     

Alterations of gyrA,gyrB, and parC and Activity of Efflux Pump in Fluoroquinolone-resistant Acinetobacter baumannii

Objectives

This study investigated the fluoroquinolone-resistant mechanism of 56 clinical cases of A baumannii infection from 23 non-tertiary hospitals, collected between 2004 and 2006.

Methods

Susceptibility testing was performed by broth microdilution and Epsilometer test. Analyses of quinolone resistance-determining region (QRDR) were done by sequencing. The activity of the efflux pump was measured using inhibitors.

Results

The sequences from selected 56 isolates were divided into seven groups (I-VII) on the basis of mutations in gyrA (S83L), parC (S80L, S80W and S84K) and gyrB (containing the novel mutations E679D, D644Y and A677V). The 27 isolates with triple mutations in gyrA, gyrB and parC (groups IV-VII) showed higher levels of resistance to ciprofloxacin (minimal inhibitory concentration [MIC] of 16-256 μg/mL) than the 26 isolates with double mutations in gyrA and parC (groups II and III, MIC of 8-64 μ g/mL; p < 0.05). Alterations in the efflux pump were observed in four isolates with the parC S80L mutation (group II) or E84K mutation (group VII), but no effect was observed in an isolate with the parC S80 W mutation (group III).

Conclusion

These results suggest that triple mutations in clinical isolates of A baumannii contribute to the development of high levels of resistance to fluoroquinolones and that mutations in parC S80L or E84K (groups II and VII) may contribute to alterations in efflux pump activity in A baumannii.     

Assessment of Antisecretory,Gastroprotective, and In-vitro Antacid Potential of Daucus carota in Experimental Rats

Objectives

In Indo China, carrots have been reported to regulate the functions of the stomach and intestines. The objective of the present investigation was to unravel the therapeutic potential of 50% ethanol extract from Daucus carota roots (EDC) on antisecretory, gastroprotective, and in vitro antacid capacity using experimental rats.

Methods

Assessment of EDC antisecretory and in vivo antacid capacities was carried out using a pyloric ligation induced ulcer model. The gastroprotective effect was assessed with an absolute ethanol induced ulcer model. The integrity of gastric mucosa was evaluated using the estimation of glutathione and gastric mucus level and with histopathological examination of gastric mucosal cells. The in-vitro antacid capacity was evaluated using a titration method. The effect of the extract on the liver was assessed by measuring serum biochemical parameters.

Results

The EDC significantly (p < 0.01–0.001) reduced gastric lesions in both models. Furthermore, the EDC also significantly (p < 0.05–0.001) reduced the volume of gastric content whereas the total acidity was significantly (p < 0.05–0.001) reduced with the doses of 100 mg/kg and 200 mg/kg EDC. Moreover, the mucus content and glutathione level increased significantly (p < 0.05) in the absolute alcohol-induced ulcer. The EDC also showed in-vitro antacid capacity. Histopathological studies further confirmed the potential of EDC by inhibiting congestion, edema, hemorrhage, and necrosis in gastric mucosa.

Conclusion

The EDC exerted antisecretory, gastroprotective, and in vitro antacid potential. These activities could be attributed due to the presence of glycosides, phenolics, tannins, alkaloids, and flavonoids.     

Comparison of Proteome Profiling of Two Sensitive and Resistant Field Iranian Isolates of Leishmania major to Glucantime? by 2- Dimensional Electrophoresis

Background:

In this study, two-dimensional gel electrophoresis (2-DE) method was applied to determine and compare the protein spots expressed in the two field isolates of Leishmania major and recovered from the patients who were clinically sensitive and resistant to Glucantime® treatment.

Methods:

Leishmania parasites were isolated from the cutaneous lesions of two CL infected patients in Shiraz, south of Iran. The species of the two isolates were identified as L. major using Nested-PCR. Sensitivity (Sh-214S) and resistance (Sh-120R) of the two isolates to meglumine antimonite were checked by the standard in vitro assays. Both sensitive and resistant L. major isolates were harvested in RPMI 1640 medium. Protein extractions were performed using TCA/Acetone method and the protein spots were determined by a two-dimensional gel electrophoresis (2-DE). The gels were stained with silver nitrate and analyzed by Image Master 2D Melanie-6 software.

Results:

About 2967 protein spots were detected. Overall, 89 protein spots represented considerable changes of expression in the resistant isolate of L. major compared to the sensitive isolate. Of these, 60 and 29 protein spots were up-and down regulated, respectively. In addition, 11 protein spots present in the resistant isolate were noticed to be absent in the sensitive isolate.

Conclusion:

A number of proteins showed significant changes of expression in the drug-resistant L. major; moreover, the roles of these proteins probably enhanced the parasite resistance to the drug and increased parasite survival in the cells.     

Characterization of Plasmid-Mediated AmpC and Carbapenemases among Iranain Nosocomial Isolates of Klebsiella pneumoniae Using Phenotyping and Genotyping Methods

Objectives

Plasmid-mediated AmpC β-lactamases (PMABLs) and carbapenemases are emerging groups of antimicrobial-resistance determinants. The aims of the study were to evaluate the occurrence of PMABLs and carbapenemases in clinical isolates of Klebsiella pneumoniae and compare the test performance of various phenotypic methods for detection of these enzymes in Iran.

Methods

A total of 100 K. pneumoniae isolates were collected from clinical specimens obtained in Valiasr Hospital. AmpC production in all isolates was determined using the AmpC disk test, the cephamycin Hodge test, the AmpC Etest, and the boronic acid combined-disk test. In addition, carbapenemase production was determined using the modified Hodge test, the EDTA disk synergy test, and the boronic acid combined-disk test. The performances of various phenotypic methods were evaluated by the comparison of their results with polymerase chain reaction (PCR) method as the gold standard.

Results

Of the 100 isolates, 19 (19%) were demonstrated to harbor the PMABL-resistance gene by the multiplex PCR method. The PCR result indicated the presence of carbapenemase genes in 12 isolates. The performance of various phenotypic tests carried out for detection of carbapenemase-producing isolates varied widely, ranging in sensitivity from 30% to 100% and in specificity from 90.8% to 100%.

Conclusion

This is the first report of MOX-type AmpC β-lactamase and bla_GES in K. pneumoniae in Iran. A comparison of the phenotypic methods showed that a combination of cefoxitin plus boronic acid is optimal for detecting plasmid-mediated AmpC enzymes in K. pneumoniae, whereas the implementation of molecular methods is often complex, requires specially trained personnel, and is associated with higher costs.     

Pathogenic Assays of Acanthamoeba Belonging to the T4 Genotype

Background

Acanthamoeba genus is introduced as opportunistic and cosmopolitan parasite. Monkey and wistar rat are appropriate models for experimental study on Acanthamoeba infection. In this study Acanthamoeba spp. were isolated from hot spring (HS), windows dust (WD) and a corneal sample of keratitis patient (KP) and their pathogenicity surveyed by in vitro and in vivo tests.

Methods

Isolates of Acanthamoeba were cultivated axenically for 12 months in PYG medium. Overall, 30 wistar rats, in 6 equal groups were used for developing experimental Acanthamoeba keratitis (AK) and Granulomatous Amoebic Encephalitis (GAE). The Keratitis and Granulomatous Encephalitis experiments were performed by intrastromal and intranasal inoculation of Acanthamoeba cysts, respectively. Pathogenicity of the three isolates was also evaluated by in vitro test using osmotolerance and temperature tolerance assays. Identification of genotypes were performed by PCR technique and sequencing.

Result

None of the isolates could perform AK and GAE in wistar rats, although all isolates were described as T4 genotype. Isolates obtained from KP and WD could grow only in 30 °C, but not in 37 °C and 40 °C. On the other hand, HS isolate grew in 30 °C and 37 °C but not in 40 °C. Moreover, all of isolate grew in 0.5 M mannitol but not in 1 M and 1.5 M.

Conclusion

T4 isolates with a long-term axenic culture and different factors related to host and parasite may play role in pathogenicity of these free-living amoebae.     

Molecular Characterization of Eimeria Species Naturally Infecting Egyptian Baldi Chickens

Background:

Coccidiosis is a serious protozoal disease of poultry. The identification of Eimeria species has important implications for diagnosis and control as well as for epidemiology. The molecular characterization of Eimeria species infecting Egyptian baladi chickens was investigated.

Methods:

Eimeria species oocysts were harvested from intestines of naturally infected Egyptian baldi chickens. The morphometry characterization of oocysts along with COCCIMORPH software was done. The DNA was extracted initially by freezing and thawing then the prepared samples was subjected to commercial DNA kits. The DNA products were analyzed through conventional polymerase chain reaction by using amplified region (SCAR) marker.

Results:

The PCR results confirmed the presence of 7 Eimeria species in the examined fecal samples of Egyptian baldi breed with their specific ampilicon sizes being E. acervulina (811bp), E. brunette (626bp), E. tenella (539bp), E. maxima (272bp), E. necatrix (200bp), E. mitis (327bp) and E. praecopx (354bp). A sequencing of the two most predominant species of Eimeria was done, on E. tenella and E. máxima. Analysis of the obtained sequences revealed high identities 99% between Egyptian isolates and the reference one. Similarly, E. maxima isolated from Egyptian baldi chickens showed 98% nucleotide identities with the reference strain. Only single nucleotide substitution was observed among the Egyptian E. tenella isolates (A181G) when compared to the reference one. The Egyptian isolates acquired 4 unique mutations (A68T, C164T, G190A and C227G) in compared with the reference sequence.

Conclusion:

This is the first time to identify the 7 species of Eimeria from Egyptian baladi chickens.     

High Prevalence of AmpC β-Lactamases in Clinical Isolates of Escherichia coli in Ilam,Iran

Objectives

Widespread use of β-lactam antibiotics could cause resistance to this group of antibiotics in pathogenic bacteria through the production of the enzyme β-lactamases. The aim of this study is to determine the molecular detection of AmpC β-lactamases among clinical Escherichia coli isolated from Ilam hospitals in Ilam, Iran.

Methods

One hundred and twelve clinical isolates of E. coli were collected from hospitalized patients and were identified by biochemical tests. They were evaluated for extended spectrum beta-lactamases (ESBLs) production, and the positive strains were subjected to AmpC enzymes; for detection of AmpC cluster genes, multiplex polymerase chain reaction was applied.

Results

The analysis showed 62.5% of isolates were ESBLs positive and that five strains revealed the AmpC cluster genes. This is the first report of FOXM cluster genes in E. coli in Iran.

Conclusion

Based on our results, the prevalence of AmpC β-lactamases is increasing in Iran, which caused failure in antibiotic therapy. So, the current study recommended the revision of antibiotic policy in Iranian hospitals.     

High Prevalence of Class 1 to 3 Integrons Among Multidrug-Resistant Diarrheagenic Escherichia coli in Southwest of Iran

Objectives

Horizontal transfer of integrons is one of the important factors that can contribute to the occurrence of multidrug-resistant (MDR) bacteria. This study aimed to determine the prevalence of integrons among MDR Escherichia coli strains isolated from stool specimens and investigate the associations between the existence of integrons and MDR properties in the southwest of Iran.

Methods

There were 164 E. coli strains isolated from January 2012 to June 2012. Fecal specimens identified as E. coli by the conventional methods. Subsequently the antibiotic resistance was assessed using Clinical and Laboratory Standard Institute criteria. The presence of class 1–3 integrons and embedded gene cassettes was verified using specific primers by multiplex polymerase chain reaction assay.

Results

Among a total of 164 studied samples, 69 (42.07%) isolates were multidrug resistant. Class 1 and class 2 integrons were present in 78.26% and 76.81% MDR isolates, respectively. For the first time in Iran, class 3 integron was observed in 26.09% MDR isolates. Significant correlations were identified between: class 1 integron and resistance to amikacin, gentamicin, chloramphenicol, ampicillin, tetracycline, nalidixic acid, and co-trimoxazole; class 2 integron and resistance to aminoglycosides, co-trimoxazole, cefalexin, ampicillin, and chloramphenicol; and class 3 integron and resistance to gentamicin, kanamycin, and streptomycin.

Conclusion

Our results indicate that integrons are common among MDR isolates and they can be used as a marker for the identification of MDR isolates. Therefore, due to the possibility of a widespread outbreak of MDR isolates, molecular surveillance and sequencing of the integrons in other parts of the country is recommended.     

Axenic Cultivation and Pathogenic Assays of Acanthamoeba Strains Using Physical Parameters

Background

The main goal of the present study was to set up an axenic cultivation of Acanthamoeba and assess the pathogenic ability of T4 genotypes from different clinical and environmental strains of Acanthamoeba using two physical assays.

Methods

Sixteen Acanthamoeba isolates including 10 environmental and 6 clinical strains were cultured axenically. Axenic cultivation was performed using Proteosepepton, yeast extract and glucose medium and TY-I-S33culture. Pathogenic survey was done using osmotolerance and thermotolerance assay. Briefly, differentosmolarity (0.5 M and 1 M) of non-nutrient agar plates were performed. One hundred fifty µl of axenic culture were collected and were inoculated in 1% agar medium. For thermotolerance assay 150 µl of amoebas from axenic culture were divided into fresh culture mediums. Cultures were incubated at 37°C and 42 °C. All plates were monitored for 24 h, 48 h and 72 h.

Results

Overall, 16 strains of Acanthamoeba isolates previously genotyped as T4 were cultivated axenically after several months. Thermotolerance and osmotolerance assay revealed that all of clinical strains, soil and animal feces strains were highly pathogenic isolates. Two dust and water strains did not grow at high temperature (42 °C) and osmolarity (1.5 M) and thus they were classified as weak pathogens.

Conclusion

Most of T4 genotypes are highly pathogenic organisms. This is an important finding since Acanthamoeba belonging to T4 type is the predominate genotype in environmental and clinical samples. The presence of highly pathogenic Acanthamoeba may pose a risk within susceptible people.     

Whole-exome sequencing reveals genetic variants associated with chronic kidney disease characterized by tubulointerstitial damages in North Central Region,Sri Lanka

Objectives

The familial clustering observed in chronic kidney disease of uncertain etiology (CKDu) characterized by tubulointerstitial damages in the North Central Region of Sri Lanka strongly suggests the involvement of genetic factors in its pathogenesis. The objective of the present study is to use whole-exome sequencing to identify the genetic variants associated with CKDu.

Methods

Whole-exome sequencing of eight CKDu cases and eight controls was performed, followed by direct sequencing of candidate loci in 301 CKDu cases and 276 controls.

Results

Association study revealed rs34970857 (c.658G > A/p.V220M) located in the KCNA10 gene encoding a voltage-gated K channel as the most promising SNP with the highest odds ratio of 1.74. Four rare variants were identified in gene encoding Laminin beta2 (LAMB2) which is known to cause congenital nephrotic syndrome. Three out of four variants in LAMB2 were novel variants found exclusively in cases.

Conclusion

Genetic investigations provide strong evidence on the presence of genetic susceptibility for CKDu. Possibility of presence of several rare variants associated with CKDu in this population is also suggested.     

Comparison of Resistant and Susceptible Strains of Trichomons vaginalis to Metronidazole Using PCR Method

Background

Metronidazole is drug of choice recommended by WHO for treatment of trichomoniasis, however, some reports claims drug resistance in Trichomonas vaginalis isolates recently. The objective of this study was to determine the minimum lethal concentration (MLC) of metronidazole in resistant and sensitive strains, as well as genetic patterns of these stains by PCR method.

Methods

From February 2006 to March 2007, in a cross sectional study, clinical and wet mount examination of vaginal smear along with culture were performed on 683 women attending to public and private outpatient clinics in Hamadan. Trichomoniasis marked based on major clinical symptoms. Diagnosis confirmed using wet mount microscopically and culture in Diamond medium. A serial concentration of metronidazole was provided and all isolated Trichomonas strains (resistant and sensitive) tested by standard method. Finally, all sensitive and resistant strains examined by PCR technique.

Results

Only 15/683, (2.2%) of patients clinically diagnosed trichomonal vaginitis were positive for T. vaginalis by wet smear and culture. The minimum lethal concentration (MLC) for clinically sensitive isolates was 25 µg/ml; however, this concentration for resistant isolates was 200 µg/ml after 24 h and 100 µg/ml after 50 h. The results of PCR examination of DNA from sensitive and resistant isolates had same pattern. The lanes appeared by two primers were 98 bp and 261 bp for both clinically sensitive and resistant strains.

Conclusion

Resistance to metronidazole in T. vaginalis has not relation to genetic variations and might be related to some physiologic pathways of organism.     

Phylogenetic Location of the Spirometra Sparganum Isolates from China,Based on Sequences of 28S rDNA D1

Background

The phylogenetic location of Chinese Spirometra sparganum isolates remains unclear. The aim of this study was to explore the phylogenetic location of the Spirometra sparganum isolates from China.

Methods

The 28S ribosomal DNA (rDNA) D1 sequences of 14 Spirometra sparganum isolates collected from thirteen locations in China were analyzed by using Neighbor-Joining (NJ), maximum parsimony (MP) and Bayesian inference (BI), respectively. To investigate the deep variance of 28S rDNA D1 region among included species, the secondary structure of 28S rDNA D1 region was also calculated using the program RNA structure.

Results

The genus Spirometra as a monophyletic group was evidenced by two inference methods (MP and BI). All sequences within the genus Spirometra had a bulge of a cytosine residue (Bulge C) in the stem 13 of the secondary structure model of 28S rRNA D1 region. Varietal sites in sequences from all thirteen Chinese isolates were appeared in loops. In loops, adenine was the most abundant base (averagely 41.9%) followed by guanine (averagely 30.0%), and cytosine (averagely 15.1%). In stems, the average percentage of G + C (58.3%) was higher than the percentage of A + T (41.7%).

Conclusion

The ‘Bulge C’ in the stem 13 of the 28S rDNA D1 secondary structure could be as a suitable mark to identify the Spirometra species.     

Methicillin-resistant Staphylococcus aureus among a network of French private-sector community-based-medical laboratories

Objective

The aim of this study was to estimate the frequency of methicillin-resistant Staphylococcus aureus (MRSA) strains in the French community and the proportion of Panton-Valentine (PVL)-MRSA.

Design

A cross-sectional study was made during a 3-month period in 2003 through a network of private-sector, community-based medical laboratories selected throughout France: the Labville network. Each MRSA isolate was included and characterized by French National Reference Center for Staphylococci. The total number of S. aureus isolates was also collected.

Results

Among the 283 patients infected or colonized by MRSA, 166 (59%) were considered as healthcare-associated, 14 (5%) as nursing-associated and 39 (14%) as community-acquired. The proportion of methicillin resistance among S. aureus was 14%. Taking into account the sampling design, the incidence of MRSA cases in French outpatients was estimated to be 0.50 [CI95%: 0.41–0.60] per 10,000 inhabitants. The molecular analysis confirmed that 80.6% belong to the Lyon clone, the most prevalent hospital MRSA clone spreading in France and 10.6% to a closely related clone. An emerging MRSA clone containing the tst1 gene was detected in six patients and the PVL-positive ST80 clone only in one, 22-year-old, patient.

Conclusion

Most of MRSA cases diagnosed in the community in France, in 2003, were elderly with specific risk factors and harbored hospital strains. The prevalence of PVL-MRSA remained low.     

Cadmium Alters the Biotransformation of Carcinogenic Aromatic Amines by Arylamine N-Acetyltransferase Xenobiotic-Metabolizing Enzymes: Molecular,Cellular, and in Vivo Studies

Background

Cadmium (Cd) is a carcinogenic heavy metal of environmental concern. Exposure to both Cd and carcinogenic organic compounds, such as polycyclic aromatic hydrocarbons or aromatic amines (AAs), is a common environmental problem. Human arylamine N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes that play a key role in the biotransformation of AA carcinogens. Changes in NAT activity have long been associated with variations in susceptibility to different cancers in relation with exposure to certain AAs.

Objective

We explored the possible interactions between Cd and the NAT-dependent biotransformation of carcinogenic AAs.

Methods

We exposed purified enzymes, lung epithelial cells, and mouse models to Cd and subsequently analyzed NAT-dependent metabolism of AAs.

Results

We found that Cd, at biologically relevant concentrations, impairs the NAT-dependent acetylation of carcinogenic AAs such as 2-aminofluorene (2-AF) in lung epithelial cells. NAT activity was strongly impaired in the tissues of mice exposed to Cd. Accordingly, mice exposed to Cd and 2-AF displayed altered in vivo toxicokinetics with a significant decrease (~ 50%) in acetylated 2-AF in plasma. We found that human NAT1 was rapidly and irreversibly inhibited by Cd [median inhibitory concentration (IC₅₀) ≈ 55 nM; rate inhibition constant (k_inact) = 5 × 10⁴ M⁻¹ · sec⁻¹], with results of acetyl coenzyme A (acetyl-CoA) protection assays indicating that Cd-mediated inhibition was due to the reaction of metal with the active-site cysteine residue of the enzyme. We found similar results for human NAT2, although this isoform was less sensitive to inactivation (IC₅₀ ≈ 1 μM; k_inact = 1 × 10⁴ M⁻¹ · sec⁻¹).

Conclusions

Our data suggest that Cd can alter the metabolism of carcinogenic AAs through the impairment of the NAT-dependent pathway, which may have important toxicological consequences.