Genotypic detection of enterotoxin producing staphylococcus aureus in food

Staphylococcus aureus is one of the most commonly identified bacteria that cause food poisoning by virtue of its variety of enterotoxins, Nasal and hand carriage of enterotoxigenic Staphylococcus aureus is an important source of staphylococcal food contamination in restaurants, therefore it is important to detect Staphylococcus aureus carriage among foodhandlers to prevent possible food contamination .The objective of the study was to detect prevalence of enterotoxigenic strains of staphylococcus aureus among food handlers in Zagazig City and to evaluate phenotypic [SETRPLA] versus genotypic [multiplex PCR] methods for detection of the staphylococcal enterotoxins. Two hundred and seventy swabs of [nose, hand and axilla] were collected randomly from 90 food workers; 3 swabs from each food worker. The swabs were inoculated on Mannitol Salt agar an incubated aerobically for 24 hours. Any suspected Staphylococcus aureus colonies were systematically identified according to standard methods. Staphylococcus aureus isolates were subjected to SET-RPLA test using SET-RPLA Toxin Detection Kit for phenotypic detection of enterotoxin production and Multiplex-PCR to genetically detect enterotoxigenic strains. Carriage rate among the food handlers was 55.5% representing 50 Staphylococcal aureus isolates from 90 food handler, the proportion of contaminated samples among 270 swabs was 18.5% [50/270]. 38[76%] out of 50 isolates were found to be enterotoxigenic by SET-RPLA and 42 [84%] out of 50 staphylococcal isolates were found to be enterotoxigenic genotypically by multiplex PCR. The number of isolates was significantly higher in nasal swabs than in hand or axillary swabs [?[2] =36.87 and p=0.0000]. Using REPLA, the number of enterotoxin producing organisms was significantly higher in nasal swabs [91.2%] than in hand [38.5%] or axillary swabs [66.6%][?[2] = = 14.48 and p = 0.0007]. Type A enterotoxin was the most common type [48%] followed by type D [18%], then type C [6%] while the least type was type A+B [4%]. It also shows that type A+B was isolated only from nasal swabs [2.9%] and type D was not found in axillary swabs. Using Multiplex PCR, gene type a was the most common type [48%] followed by gene type d [22%] then gene type c [6%] while the least type was genes type a+ b [4%]. It also shows that gene types a+b and b+d were isolated only from nasal swabs [5.9% each]. Compared with multiplex PCR, specificity and positive predictive value of RPLA were 100%, while sensitivity was 90.48% and negative predictive value was 66.67%. This study concluded that nasal and hand carriage of enterotoxigenic S.aureus by food handlers is an important source of staphylococcal food contamination in restaurants and fast food outlets so it is important to detect S.aureus carriage among food handlers to prevent possible food contamination by them resulting in food poisoning.It also concluded that multiplex PCR is reliable and valuable method in detection of enterotoxigenic S.aureus strains and it is more sensitive and specific than SET-RPLA. This study recommended health education of food handlers to decrease contamination of their hands, settings training courses for food handlers to learn them the proper hand washing technique and frequent examination of food handler to find and treat. It also recommends screening food workers to identify staphylococcus aureas carriers and referring them to the appropriate health authorities for decolonization

Bacteriological and immunological study in diabetic foot

Background: Diabetes mellitus is increasing rapidly through the world, so the diabetic foot syndrome become more and more important as a major diabetic complication

Objectives: The aim of study was to determine the association between the up-regulation of circulating level ofIL-6 in diabetic patients with foot ulcer compared with diabetic patients without foot ulcer

Subjects and methods: The study included 60 subjects, they were divided into three groups; group I included 20 diabetic patients without foot ulcer syndrome, group II included 20 diabetic patients with diabetic foot ulcer syndrome [DFUS] and group HI included 20 apparently healthy subjects as a control. All subjects were subjected to clinical assessment, routine laboratory investigations and specific investigations including assay of glycosylated haemoglobin, serum IL-6 and bacteriological culture and sensitivity for ulcer

Results: There is no significant difference among the three studied groups regarding the gender, age, duration of the disease and type of treatment. There was significant difference between group I and other groups regarding hypertension [p<0.02]. There was no significant difference between the three studied groups regarding WBC. There was a significant difference between the three studied groups as regards neutrophils and platelets counts f [p=0.009] nd [p=0.03] respectively. There was a highly significant difference between the three studied groups as regards Hb concentration [p<0.001]. There was a highly significant difference [p<0.00l] between group II and both group I and group III as regards ESR [43 +/- 77.2, 13+/-3.9, 11+/-3], random blood sugar [RBS] [319.7+/-47, 238+/-47.8, 92.5+/-10.8], glycosylated haemoglobin [HbAlc] [9.93+/-1.35, 8.83+/-1.4, 4.6+/-0.6], Urea [49.9+/-37, 52.5+/-41, 20.6+/-2.4] and IL-6 [18.9+/-5.6, 4.9+/-2.7, 2,77+/-I].There was positive significant correlations [p<0.001] between IL-6 and levels of RBS [r=0.72], and HbAlc [r=0.62], respectively. Also, a positive significant correlation between IL-6 and neutrophils% [r=0.35, p<0.005] was found The most common isolated microorganisms from foot culture were mixed gram + ve cocci and gram -ve bacilli representing 60% and lonely gram + ve cocci and gram -ve bacilli were 20% respectively. Also, it was found that the most gram +ve organism was Staphylococcus aureus and the most gram -ve organism was E. coll and the most effective antibiotic was Ampicillin-Sulbactam 70% followed by Imipenem 30%

Conclusion: Diabetic patients with foot ulcer were found to have higher IL-6 level than diabetic patients without foot ulcer and they were prone to complications or mortality. This assay could facilitate early and accurate diagnosis and greatly aid timely institution of appropriate treatment

Helicobacter pylori Infection and its response to treatment in diabetic Patients versus non diabetics

Background: H. pylori has been recognized as a public health problem worldwide affecting approximately 50% of the world population and more prevalent in developing than the developed countries. It is a common infection in diabetic patients

Aim of the work: The study designed to study the prevalence of H. pylori infection in diabetic patients in comparison with non diabetic patients and the response to treatment ofH. pylori in both groups

Methods: WO patients were enrolled in the study. They were classified into 2 groups: Group [I] 50 patients complaining of dyspepsia with DM, and group [2] 50 non-diabetic patients complaining of dyspepsia. Serum H.pylori IgG antibodies and stools H.pylori antigen were determined for all patients by Gen Way H. pylori IgG ELISA and OneSite H pylori rapid test respectively

Results: H. Pylori stools Ag was positive in 58% of diabetics and 40% of non diabetics with no significant difference [P value 0.085]. Serum H.pylori Ig G were significantly positive in diabetes [52%] compared to non diabetic patients [32%]; P value 0.05. HE Ale was significantly higher in diabetics positive H.pylori cases compared to diabetics negative for H.pylori. Change in the HBAlc blood level before and after H.pylori treatment was highly significant in diabetic cases [P value <0.001]

H.pylori was more resistant to treatment in diabetic patients compared to non diabetics

Conclusion: No significant difference between both diabetics and non diabetics regarding prevalence of H pylori infection. Type 2 diabetic patients showed a significantly lower eradication rate than non diabetic patients. Successful eradication of H pylori infection significant I improve HBAlc

Phenotype and genotype of some clinical group B streptococcus isolates resistant to erythromycin in Egypt

The aim of the study was to determine susceptibility of group B streptococci [GBS] to commonly used antibiotics and to determine the phenotype of macrolide resistance correlating it with the genotype. 220 group B streptococcus strains were subjected to antibiotic sensitivity disc diffusion method to penicillin, ampicillin, ceftriaxone, tetracyclines, erythromycin [E] and clindamycin [CD]. Erythromycin resistance phenotype was done by double disc diffusion test. Erythromycin resistance mefA, ermB, and ermTR genes were determined using multiplex PCR. The results revealed that all strains were sensitive to penicillin, ceftriaxone and ampcillin. 161 [73%], 38 [17%] and 24 [11%] strains were resistant to tetracyclines, erythromycin and clindamycin respectively. 15 [39.5%] were resistant to E alone [M phenotype]. M phenotype strains were associated with mefA gene. 23 [60.5%] isolates were resistant to both E and clindamycin [CD]. 9 [23.7%] strains were inducibly resistant to clindamycin [iMLSB] and all had ermTR gene.. 14 [36.8%] were constitutively resistant to clindamycin [cMLSB]. cMLSB strains have different genotypes. 7 strains were ermB genotype. 4 strains were ermTR genotype. 2 strains had both ermB and ermTR genes while only one strain had both ermB and mefA genes. Only 1 strain was resistant to clindamycin but sensitive to E. MefA was detected in 16 [42%] E resistant strains. ErmTR was also detected in 16 [42%] E resistant strains. ErmB was detected in 10 [26%] E resistant strains. 31[82%] E resistant strains contain single resistance gene while 7[18%] resistant strains contain more than one resistance gene. The study findings conclude that GBS isolates remain uniformly susceptible to penicillin and ampicillin but he erythromycin resistance has reached substantial level. The study recommends that testing of susceptibility to erythromycin and clindamycin by double disc diffusion method should be performed in individual cases when considered as alternatives for prophylaxis and treatment of GBS infection or colonization because strains with inducible MLSB cannot be detected with the conventional disc diffusion method

Slime forming staphylococcus epidermidis isolated from orthopedic prosthesis infections and its sensitivity to antibiotics

Staphylococcus epidermidis [S.epidermidis] is a frequent cause of infections of indwelling medical devices especially those with orthopedic implants. S.epidermidis grows on medical devices as an adherent biofilm consisting of cells enmeshed in a sticky, extracelluar slime that is firmly attached to the underlying surface. The slim matrix makes S.epidermidis biofilm highly resistant to antibiotics and host defenses and nearly impossible to eradicate. The aim of the study is to determine importance of slime formation in S. epidermidis orthopedic prosthesis infections and to investigate if slime formation has an effect on its antibiotics sensitivity. 80 coagulase negative staphylococcus strains [CoNS] were isolated from 200 tissue specimens of patients with orthopedic prothesis infections. Out of these 80 CoNS, 52 [65%] strains were S.epidermidis. Isolated S. epidermidis were plated on Congo red agar and subjected to PCR to detect icaA and icaD genes to identify and confirm slime producing strains respectively. All biofilm producing strains were subjected to MIC and MBEC using Calgary Biofilm Device[CBD]. 36 [69%] S. epidermidis strains were slime [biofilm] producers and 16 [31%]strains were non slime [non biofilm] producers by CRA, while by PCR 39[75%] strains of S. epidermidis were biofilm producers and 13 [25%] strains were non biofilm producers. The results also revealed that the minimal biofilm eradication concentrations [MBECs] were higher than the corresponding conventionally determined MICs for all antibiotics tested. MIC 50 and MBEC 50 for vancomycin, were 2 micro g/ml versus 8 micro g/ml, gentamycin, 1 micro g/ml versus 32 micro g/ml, oxacillin, 4 micro g/ml versus 16 micro g/ml, erythromycin, 8 micro g/ml versus 64 micro g/ml, ciprofloxacin, 0.5 micro g/ml versus 2 micro g/ml and cephalothin 4 micro g/ml versus 16 micro g/ml. MIC90 and MBEC90 for vancomycin were 4 micro g/ml versus16 micro g/ml, gentamycin, 16 micro g/ml versus 128 micro g/ml, oxacillin, 8 micro g/ml versus 128 micro g/ml, erythromycin, 16 micro g/ml versus 128 micro g/ml, ciprofloxacin, 4 micro g/ml versus 8 micro g/ml and cephalothin 32 micro g/ml and 128 micro g/ml. The results of the present study confirm that ica genes can be considered a virulence marker in the pathogenesis of implant associated orthopedic infection by S. epidermidis. This study also demonstrates marked differences between the results of susceptibility testing performed according to standard NCCLS guidelines and testing based on biofilm susceptibility testing

Assessment of T helper 1 and T helper 2 cytokines and their correlations with viral load in HCV patients co-infected with S. mansoni

The aim of this study was to investigate the effect of infection with S. mansoni on the balance between Th-1 and Th-2 cytokines in patients with chronic hepatitis C virus [HCV] infection and to show the relations between these two groups of cytokines to the degrees of viral load in these patients. 44 individuals were classified into 4 groups: group I of control subjects [n=10], group II of patients with S. mansoni mono-infection [n=9], group III of patients with chronic HCV mono-infection [n=13] and group IV of patients with both S. mansoni and HCV co-infection [n=12]. For individuals of all studied groups, interferon-gamma and IL-2 [cytokines of Th-1 cells] and IL-4 and IL-10 [cytokines of Th-2 cells] were measured. Viral load was measured for patients of group III [HCV mono-infection] and group IV [co-infected patients]. The results showed that Th-1 cytokines [IFN-gamma and IL-2] were significantly higher in HCV mono-infection patients and significantly lower in patients co-infected with HCV and S. mansoni compared to normal subjects group. In S. mansoni mono-infection group, IFN-gamma was decreased while IL-2 was normal compared to normal control group. Th-2 cytokines [IL-4 and IL-10] were significantly higher in the three patients groups compared to the control group but the degree of increase of IL-4 showed no significant difference between S. mansoni mono-infection patients and HCV mono-infection patients while the degree of increase of IL-10 was higher in S. mansoni mono-infection patients compared to HCV monoinfection patients. The degree of increase of both IL-4 and IL-10 was significantly higher in the coinfection patients compared to the HCV mono-infection patients. Viral load was significantly higher in the co-infected group compared to HCV mono-infection group and in both groups, the viral load was positively correlated with Th-2 cytokines and negatively correlated with Th-1 cytokines [except IL-2 in the group of HCV mono-infection patients]. In conclusion, these results suggest that HCV patients co-infected with S. mansoni suffer from strong activation of Th-2 cells and so increase of Th-2 cytokines that suppress Th-1 cells and related cytokines which is the type of immune response needed in face of HCV. This Th-1/Th-2 imbalance allows more viral replication and higher viral load in these patients compared to HCV patients who are not co-infected with S. mansoni and this may give an explanation to the rapid hepatic deterioration of these co-infected patients

Rapid diagnosis and characterization of diarrheagenic Escherichia coli in Egyptian children using multiplex PCR

Escherichia coli [E.coli] is the most important etiologic agent of childhood diarrhea and represents a major public health problem in developing countries. Aim of this work was to investigate the role of diarrheagenic E.coli in Egyptian children below 5 years age using multiplex PCR and to evaluate multiplex PCR in rapid diagnosis of enteric infections caused by diarrheagenic E.coli strains. Rectal swabs were taken from 83 children under 5 years age with diarrhea and 33 age-matched controls. All E.coli isolates were O serotyped using E.coli O polyvalent and monovalent antisera and subjected to multiplex PCR assay with specific primers, eae primer of eaeA [gene of intimin of EHEC and EPEC], primer bfpA of bfpA [structural gene for the bundle-forming pilus of EPEC], primers VT1 and VT2 of vt1 and vt2 genes [genes of shiga toxins 1 and 2 of EHEC respectively], primer LT of eltB [gene of labile toxin of ETEC], primer ST for estA [gene of stable toxin of ETEC], primer SHIG of ial [invasion-associated locus of the invasion plasmid found in EIEC] and primer EA of pCVD [the nucleotide sequence of the EcoRIPstI DNA fragment of pCVD432 of EAEC]. The study revealed that diarrheagenic E.coli strains were significantly isolated from patients more than control using multiplex PCR. Out of 70 E.coli strains isolated from patients, 17[24.3%] isolates were proved to be diarrheagenic by multiplex PCR where 53 [75.7%] isolates were non diarrheagenic. Out of 30 E.coli isolates recovered from control group, 1 [3.3%] isolate was proved to be diarrheagenic by multiplex PCR where 29 [96.7%] isolates were non diarrheagenic[Chi-square=18.5 and p

Interleukin-1 receptor antagonist among chronic renal failure pediatric patients with and without hemodialysis

During hemodialysis [HD], human blood leucocytes in circulation are exposed to several extraneous challenges, thus stimulated to secrete many inflammatory cytokines and its inhibitors as inter-leukin-1 and interieukin-1 receptor antagonist [IL-Ira]. The aim of this study was to investigate difference in the level of IL-IRa synthesis by peripheral blood mononuclear cells [PBMC] between CRF patients under conservative treatment and CRF patients under hemodialysis, and if this cytokine-specific inhibitory protein of PBMC can be used as a marker of dialysis related morbidity. The study included 44 subjects divided into 3 groups: [A] control group, [B] Chronic renal failure[CRF] under conservative treatment, [C] CRF under HD. Peripheral blood mononuclear cells [PBMC] were separated by ficoll-Hypaque. Spontaneous and Phytohemagglutinin [PHA] stimulated total IL-1Ra synthesis [cell-associated and secreted] by cultured PBMC was measured using EL-ISA method. The results of the study revealed that there were significant spontaneous total IL-IRa synthesis by PBMC in dialysis patients [group C] 2812 +/- 836 and in patients with conservative treatment [group B] 1791.2 +/- 252 compared to control group [group A] 940 +/- 227.8 [P value <0.001]. There were significant spontaneous total IL-IRa synthesis by PBMC in dialysis patients [group C] 2812 +/- 836 compared to patients with conservative treatment [group B] 1791.2 +/- 252 [P value <0.001]. There were significant PHA stimulated total IL-IRa synthesis by PBMC in dialysis patients [group C] 34041 +/- 8906 and in patients with conservative treatment [group B] 8565 +/- 1244 compared to control group [group A] 2980 +/- 608 [P value <0.001]. There were significant PHA stimulated total IL-IRa synthesis by PBMC in dialysis patients [group C] 34041 +/- 8906 compared to patients with conservative treatment [group B] 8565 +/- 1244 [P value <0.001]. There was positive correlation between PHA stimulated total synthesis of ILIRa and spontaneous total synthesis of ILIRa by PBMC in the 3 groups [P value <0.001]. There was significant correlation between IL-IRa [spontaneous and stimulated] with blood pressure, urea and creatinine level [P value <0.001]. This study concluded that PBMC of CRF patients synthesize significant level of IL-IRa compared to PBMC of healthy subjects. Also, PBMC of CRF patients on HD synthesize significant level of IL-IRa compared to PBMC of CRF under conservative treatment. Lastly, IL-IRa synthesis by PBMC of CRF patients was correlated with blood urea, serum creatinine and blood pressure level

study of HLA type II [DRB] by PCR in Egyptian rheumatoid arthritis patients

To assess the relationship between HLA-DRB1 and rheumatoid arthritis in Egyptian patients, 40 subjects ere examined; 26 were rheumatoid arthritis patients and 14 were normal persons. The patients were further subdivided into groups according to the Mean Grades of the Disease Activity. All patients and the controls were subjected to ESR estimation by Westgren's method, Rheumatoid factor testing and HLA-DRB1 typing by PCR. The DNA was extracted from whole blood. Exon 2 was amplified and checked by gel electrophoresis. For HLA-DRB1 allele detection, amplification products were subsequently hybridized using typing strips in which 37 sequence specific DNA probe lines and 2 control lines were fixed. There was a significant association between rheumatoid factor and the grade of disease activity. There was a significant association between HLA-DRB1 broad types *4 and *1 and the susceptibility to rheumatoid arthritis. HLA-DRB1 broad types *4 and *1 had significant higher frequencies in patients than controls. They were 40.4% and 21.2% in patients while were 10.7% and 3.6% in controls. P values were 0.04 and 0.003 respectively for them. HLA-DRB1 *0102 allele had shown a significant increase in group IV rheumatoid arthritis patients with a frequency of 50%. high relative risk, and a P value of 0.014