Acute Pyelonephritis in Adults:
Clinical and Therapeutic Features

N. Bouzouaia, M. Ben Jemaa, M. Hsairi,
T. Ben Chaabane, S. Ben Redjeb
Laboratoire " Résistance aux Antibiotiques " - Faculté de Médecine - Tunis

Introduction:
Acute pyelonephritis (AP), a severe urinary tract infection (UTI), constitutes a major health problem.

Objectives:
The aim of the study was to analyze the epidiomologic, clinical, bacteriological and therapeutic features of AP in adults with the intention of improving treatment of this UTI.
Patients and method:
The study includes 421 cases suffering from AP, hospitalized during one year, in three university infectious disease wards. Epidemiologic, clinical and bacteriological features were described. Diagnosis was confirmed by urine culture (bacteriuria 10⁵ CFU/ml) in all cases.
Data have been entered on Epi Info (version 6)and analyzed with Stata (version 6).
Results:
AP occurs mainly in young women (63.4 %). It was complicated in 63.4 % (urolithiasis (26 %), diabetes (22.8 %), elderly (22 %), genital abnormalities in males (28 %).
E. Coli was predominant in uncomplicated AP (88.4 - 88.9 %). In case of predisposing factors and recurrent AP, P. aeruginosa, Klebsiella, Proteus, Enterococcus and coagulase negative Staphylococcus were isolated.

E. Coli resistance to ampicillin was high (58 %), amoxycillin-clavulanic acid (29 %), trimethoprim-sulfamethoxazole (38 %), cephalotin (23 %). Clinical cure and microbiological eradication were obtained in 86 % and relapse was observed in 13.3 %. A single or a combined course of antibacterial agents was prescribed using first and third generation cephalosporins, ciprofloxacin, trimethoprim-sulfamethoxazole, aminoglycoside during 16.9 to 19.5 days in female and 20.9 to 27.8 in male.
Conclusion:
This epidemiologic, bacteriological and therapeutic study points out the divergence between guidelines and real management of AP in hospitals. Drafting antibiotic prescription guides explaining AP management would be helpful for improving physicians' practices. National guidelines were drawn up on April 2002 according to local epidemiology and data of antimicrobial resistance surveillance.

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Efficacy of Using Frozen Breast Milk after Thawing for Infant Feeding during Mothers' Work.
Prof . Haifa' Tawfeek,
College of Medical & Health TechnologyBaghdad, Iraq

Background: Women's can take the infants with them and breast - feed, but are often forced to take jobs where breast feeding is impossible.
Objectives: Therefore the object of the present paper is to study bacterial content of expressed breast milk.
Methods: The study involved the collection of 178 breast milk samples from lactating mothers attending MCHs centers in Baghdad area during 1998 - 2000...
All mothers received instruction on breast and hand cleansing and on the technique of manual expression of the breast before collection of the samples. Samples were obtained into sterile glass jars and were frozen at -180°C
In the laboratory, frozen samples were allowed to thaw at room temperature (22 - 25 0C). The thawed specimens were divided into three portions. One portion was stored at 4°C while the second portion was examined for degree of bacterial contamination at different intervals up to nine hours. The third portion was held in the refrigerator (4°C) for 72 hr and then examined for total bacterial content.
The method used for the analysis of milk samples were those described in the Standard Method for the Examination of Dairy Products. Samples were examined for total bacterial count, coliform count, Escherichia coli, Staphylococci and Streptococci. Results are expressed as colony forming units (CFU) per milliliter.
Results: The results revealed that the frozen milk samples showed a bacterial count not more than 3 x 10² bacteria / ml after thawing and 9 hr. Storage in the refrigerator.

Conclusion: The use of frozen breast milk for infant feeding during mother's work is safe.

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Enzyme Biovaibility and Depletion of
Slaughter- House- by- Products

M.R.A. Alldialidy, M.A. Albayaty N.Y.
College of Medicine & Health Tequnology - Baghdad, College of Veterenary Medecal Tequonology - Baghdad

The present study was carried out to utilize the by - product of slaughter houses and fish for production of enzyme and blood proteins,
The obtained results were the following :-
1-Dried by- product which were obtained from slaughter - house and local markets of Baghdad Governorate which included poultry heads and leg, fish intestine contained the highest protein content (75.49 %), where poultry legs contained the lowest protein content (44-44%) on the dry weight basis.
2-Pepsin and fryghtn were extracted from cow abornasum and intestines directly after slaughter of six cow. Pepsin was extracted and gave the highest specific activity (307.5 unit /mg protein). Trypsin was extracted by distilled water gave the highest specific activity (31.7 unit /mg protein).
3- Six bovine pancreas samples were used to extract chymotrypsin by 0.25 M sulfuric acid and the specific activity was (24.6 unit /mg protein). Panceratin powder was also prepared using cold acetone from bovine pancreas (16% of original pancreas weight).
4- Various blood protein were fractionated by different ammonium sulphate concentration where 1.4 gram fibrinogen, 2.1 gram euglobulins, 1.0 gram pseudoglobuline and 2.5 gram albumin at 25,33,46 and 64% saturation, respectively. The total amount of albumin at fractionated proteins was 7.0 gram /100 ml plasma.

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Correlation between Pulmonary Tuberculosis and Pneumococal Infection
Prof. Zuhair N. Hamad
College of Medical and Health technology Department of Clinical Pathology, Baghdad, Iraq

This study aimed to estimate the possible role of Pneumococci in pulmonary tuberculosis as well as the antagonism against other bacteria.
250 sputum samples were collected from patients with acute & chronic T.B. , patients with lung infection , and from healthy individuals.
Cultural morphological and biochemical properties were employed to identify bacterial species.
Disc method was used to study antibiogram for bacteria other than M. tuberculosis.
Agar diffusion method was used to study antagonism between bacterial isolated. Pneumococci dominated in T. B. patients, while it was a small percentage in other categories.
Multiple drug resistance was well defined among Pneumococci isolated from different categories. Pneumococci has a great potency to antagonize Staph aureus & Strept. pyogenes .
Results indicated high correlation between the exsistence of Pneumococci in patients with acute or chronic T. B.
Conclusion:
Results gave a frank sign for the possible role of resistant Pneumococci in interference with T.B. treatment regimen (DOTs).
Recommendation:
Careful genetic analysis of the relationship between Pneumococci & T. B.

Study of the nature of antagonism between Pneunococci & other bacteria.

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A Method for Testing the Antimicrobial
Susceptibility
Mohamed M. Emara
Faculty of Pharmacy , Helwan University

A rapid micro-broth-dilution assay for determining the anti-microbial susceptibility of different gram-positive and gram-negative organisms was developed. This method is based on the use of NR as a growth marker, and on the ability of viable organisms to reduce nitrates to nitrites. Bacterial viability is detected by a positive nitrite reaction rather than visible turbidity. The nitrate reduction assay was compared with standard microassay using 672 isolates of different genera, species and strains, against 30 antibiotics belong to different classes. An excellent agreement between the two methods was found (92.3%), and only (7.7%) of 11290 trials showed difference in the determined MIC by two-dilution interval above or below the turbidimetric determined MIC under the same test conditions. But the nitrate reduction assay was more rapid and sensitive in detecting viable bacteria, and so established a more accurate estimate of the minimal inhibitory concentration. The nitrate reduction assay offers the additional advantage that it could be used to determine the minimal bactericidal concentration without having to subculture the broth.. Furthermore, 623 cases of resistance were detected by nitrate reduction assay, which were not detectable by the traditional turbidimetric method.

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Regulation of Bacterial Chromosome Replication by
Response Regulator Proteins

Rania Siam, Ann Karen Brassinga and Gregory T. Marczynski

We employed Caulobacter crescentus to investigate mechanisms by which bacteria regulates chromosome replication. This bacterium is a model organism for replication as it yields two distinct progenies with different replication potentials. The asymmetric division of the predivisional cell yields both a replication competent stalked cell and a replication incompetent swarmer cell. Only in the swarmer cell is a response regulator protein cell cycle transcription regulator A (Ctr A). The swarmer cell maintains its repressed, chromosomal state by binding to five binding sites in the cloned replication origin (Cori), designated a-e. We show that phosphorylated CtrA is the active form of the protein; it binds sites [ a-e] with 35 to 100 - fold lower K(d) values than unphosphorylated CtrA. ACtr phosphorylation stimulates two distinct modes of binding to the replication origin. Phosphorylation stimulates weak intrinsic protein-protein cooperation between half - sites and does not stimulate CtrA-P binding unless protein -DNA contacts are made at both half-sites. CtrA phosphorylation also stimulates cooperative binding between complete sites [a] and [b] . However, binding to each of the other CtrA-binding sites [c] , [d] and [e] is completely independent and suggests a modular organization of replication control by CtrA. We therefore propose a model where a phosphorelay targets separate biochemical activities inside the replication origin through both cooperative and independent CtrA - binding sites (Siam and Marczynski, 2000).

We further dissect the biochemical characteristics of this response regulator protein. A CtrA aspartate to glutamate (D5IE) mutation mimics phosphorylated CtrA approximately P in vivo and rescues non- viable
C.crescentus cells. However , we observe that the CtrA D51E and the unphosphorylated CtrA wild-type proteins have identical DNA affinities and produce identical DNase I protection footprints inside the C. crescentus replication origin. Therefore, D51E promotes essential CtrA activities separate from increased DNA binding. Accordingly, we argue that CtrA protein recruitment to target DNA is not sufficient to regulate cell cycle progression . These results triggered us to further determine CtrA interaction with other replication proteins (Siam and Marczynski, 2003).

The integration host factor protein (IHF) is a known replication protein that alters the DNA architecture and folds the chromosome in distinct ways to promote protein/DNA or protein/protein interaction. We demonstrate that IHF binds Cori over the central CtrA binding site c. Surprisingly, IHF and CtrA share DNA recognition sequences. Rather than promoting cooperative binding, IHF binding hinders CtrA binding to site c and nearby site d. Unlike other CtrA binding sites, DNA mutations in the CtrA c/IHF site uniquely impair autonomous Cori plasmid replication. These mutations also alter transcription from distant promoters more than 100 bp away. When the CtrA c/IHF site was deleted from the chromosome, these cells grew slowly and became selectively intolerant to a CtrA phosphor-mimic allele (D51E) . Since CtrA protein concentration decreases during the cell cycle as IHF protein concentration increases, we propose a model in which IHF displaces CtrA in order to bend Cori and promote efficient chromosome replication (Siam et al., 2003).
CtrA is the first identified response regulator protein to bind in the replication origin and regulates chromosome replication and bacterial development. We tested if other bacterium shares a similar replication control by studying the genome arrangement and comparing them to Caulobacter crescentus genome. A
30-kb region surrounding the replication origin in Caulobacter crescentus was analyzed . Comparison to the genome sequence of another alpha-proteobacterium, Rickettsia prowazekii , revealed a conserved cluster of genes (RP001 , hemE , hemH and RP883) that overlaps the established origin of replication in C. crescentus and the putative origin of replication in R. prowazekii. The genes flanking this cluster differ between these two organisms . We therefore propose that this conserved gene cluster can be used to identify the origin of replication in other alpha-proteobacteria (Brassinga et al., 2001).
In addition a homolog of the Caulobacter crescentus global response regulator CtrA was identified in Rickettsia prowazekii . CzcR . CzcR expression partially compensates for developmental defects in CtrA mutant C. crescentus cells , and CzcR binds to all five CtrA binding sites in the C. crescentus replication origin. Conversely , CtrA binds to five similar sites in the putative R. prowazekii replication origin (oriRp). Also, Escherichia coli IHF protein binds over a central CtrA binding site in oriRp . Therefore, CtrA and IHF regulatory proteins have similar binding patterns in both replication origins, and we propose that CzcR is a global cell cycle regulator in R. prowazekii (Brassinga et al., 2002)

Two-component and phosphorelay signal -transduction systems of pathogenic bacteria control the expression of genes encoding essential functions, virulence factors and antibiotic resistance . Recent studies have confirmed the crucial role of two-component systems in the pathogenesis of diseases caused by several microorganisms and highlighted the validity of using these systems as targets for therapeutic intervention.
The emergence of strains of pathogenic bacteria that are resistant to multiple antibiotics has driven the search for new targets and/or modes of action for anti-microbial agents.
The presence of essential two-component systems in bacteria and the central role that these regulatory systems play in virulence and antibiotic resistance has meant that two-component systems and phosphorelays have been recognized as targets for antimicrobial intervention. Currently CtrA, CzcR and other response regulators are under study as target for antimicrobial therapy.

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Multidrug-Resistant Mycobacterium tuberculosis : Comparison between the Standard Susceptibility and the DNA - Based Methods

Nahed Ibrahim, Khalifa I, Ahmed A, Cooksey R, Farag I

Microbiology & Immunology Department, Faculty of Medicine, Suez Canal University and CDC , Atlanta Georgia, USA

Early detection of drug resistance in M. tuberculosis isolates is crucial for appropriate treatment to prevent the development of further resistance and the spread of resistant strains . The identification of resistance mutations enables the development of molecular test, which may result in a reduction of turnaround times for susceptibility results to 1 to 2 days. The aim of this study was to compare the agar proportion and SSCP methods for the detection of multiple drug resistance (MDR) in M. tuberculosis . A second goal was to characterize the mutations in rpoB and katG genes associated with rifampin (RIF) and isoniazid (INH) resistance by DNA sequencing . The study was conducted on 40 MTB isolates collected from smear-positive pulmonary tuberculosis patients from two major chest hospitals in Suez Canal area in Egypt. Drug susceptibility testing was performed on the MTB isolates against four anti-tuberculosis drugs (INH , RIF , SM and EMB) by the L-J agar proportion method. The highest rate of resistance was that for SM (47.5%) while the lowest was for EMB (25%) . The rate of INH resistance was 45% (18 isolates) and that for RIF was 37.5% (15 isolates). The rate of MDR (resistance to both INH and RIF) was 35% . The SSCP was performed on the 40 MTB isolates after amplifying the 321 bp region of katG and the 128 bp region of rpoB in which the most common mutations implicated in drug resistance are found . SSCP detected 13 INH resistant isolates ( 32.5%) while it detected 12 (30%) RIF resistant ones out of 40 MTB
isolates. When comparing the results of SSCP to those of the agar proportion method (the gold standard) , we found that its sensitivity was 72.2% and 80%
when testing for katG and rpoB mutations respectively ; its specificity was 100%.
The resistant isolates detected by SSCP were subjected to DNA sequencing to identify the mutations n the katG and rpoB gene regions examined. DNA sequencing of the 13 INH resistant isolates , detected by SSCP , revealed that 12 had a S315T amino acid substitution and the remaining one isolate had a D329A substitution. Of the 12 RIF - resistant isolates , shown to have mutations in rpoB by SSCP , 10 had a S531L substitution and 2 isolates had H526Y, H526L substitution respectively . It is concluded that SSCP is a rapid, relatively inexpensive and easy to perform technique. It can detect drug resistance much faster than the conventional phenotypic methods.

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Genetic Analysis of Rifampin Resistance in
Mycobacterium Tuberculosis

Gehan Galal*, Saied abbadi**, Amina Medhat***,
Mohammed Abd El Fatah***and Nabila El Sheikh****

Egyptian Holding Company for Biological Products and Vaccines (VACSERA) Department of Microbiology* , Faculty of Medicine, Suez Canal University**, Department of Biochemistry , Faculty of Science , Ain Shams University***, Department of Microbiology, Faculty of Medicine, Al Azhar University****

Mutations of the rpoB associated with rifampin resistance were studied in 25 multidrug resistant (MDR) - Mycobacterium Tuberculosis isolates . Of all isolates , 17 had a mutation in the 81bp region of the rpoB gene revealed by single strand conformation polymorphism (SSCP) and DNA sequencing . Only two resistance patterns were detected by SSCP analysis. DNA sequencing revealed that Ser531----->Leu arose most frequently missense mutation (70.5%) followed by Asp516----->Val (23.5%). A silent mutation [Gly 536 (CTG-----> GTG)] combined with the mutation Ser531-----> Leu and Leu545-----> Val , was observed in one resistant strain. The sensitivity and specificity of the SSCP was 100% compared with that of the DNA sequencing . These results suggest that SSCP is an efficacious method of predicting rifampin resistance and would reduce the time required for susceptibility testing for approximately 4-8 weeks to few days and it is useful for rapid screening of rfampin resistance in susceptible and fully resistant isolates of M. Tuberculosis.

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Resistance to Imipenem and Detection of bla_IMP
Gene in Pseudomonas aeroginosa.

Eman M. El-Behedy, Heba A. Mohtady, Elham Sami, Fatma A. Amer, Hala E. Zanfaly*, Dalal E. M. Soud*, Salem Khalil**, Yasser A. El-Hendy***, Eman El-Gendy****
Microbiology and Immunology Department, Anaesthesiology Department*, Department of Urology**, Department of Medicine***, Department of Gynecology and Obstetrics****, Faculty of Medicine, Zagazig University, Zagazig, Egypt

Imipenem (IPM) is a potent β- lactam, partly because of its resistance to hydrolysis by most β- lactamases except carbapenamases. The latter enzyme, a metallo β- lactamase belonging to molecular class B, is capable of hydrolyzing both IPM and ceftazidime (CAZ), in addition to most β- lactam antibiotics, and confers resistance to these agents in pathogenic bacteria. bla_IMP is the gene encoding the carbapenem-hydrolysing mettalo- β- lactamase. Surveillane for the identification of bla_IMP by PCR techniques revealed that the gene was disseminated among various gram-negative pathogens especially in P. aeruginosa and S. marcescens. The current work was carried out to determine the incidence of resistance to imipenem among Pseudomonas species isolated from various clinical conditions and to survey for the existence of bla_IMP among isolated strains. Pseudomonas were isolated from various clinical conditions. Their susceptibility to ampicillin, imipenem, ceftazidime, cefotaxime, , carbenicillin, piperacillin, aztreonam, amikacin, gentamicin and ciprofloxacin, were determined. Examination for the carbapenamase gene was done by PCR method. 179 strains of Pseudomonas species were isolated. 5 strains were resistant to imipenem. They were also resistant to ampicillin, carbenicillin, cefotaxime, and ceftazidime. 2 strains were resistant to piperacillin, aztreonam
and ciprofloxacin. 3 strains were resistant to gentamicin and 4 strains were resistant to amikacin. bla_IMP was detected by PCR in the five strains. We conclude that the incidence of resistance to imipenem is low, however, rational use of this drug is indicated to avoid the increase in that incidence.

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Plasmid Profile of Multidrug Resistant Pseudomonas
Strains in a Burn Unit

Bothina A.Koura, Mohammed . H .Eldeen Zaghloul1, Noha El Mashad*
Department of Microbiology, Faculty of Medicine, Menofiya University,Department of Clinical Pathology*, Faculty of Medicine, Mansoura University, Egypt.

Pseudomonas aeruginosa is an important nosocomial pathogen , especially in individuals who are immunocompromised . This study was
performed to study the epidmiology of pseudomonal infections in Burn Wound
Centre , Menoufiya University Hospital . Pseudomonas isolates were identified
to the species level using automatic Sensititer , subjected to antibiotic
susceptibility testing by the disc diffusion and agar dilution methods , β
lactamase production was evaluated using iodometric method, extended
specttrum β- lactamase production were tested using double-disk clavulanate
synergy test . Plasmid profile analysis by agarose gel electrophoresis
was performed . Results showed that bacterial microorganisms were isolated
from 100% of septic burn wound , 92 isolates were identified from 62
patients . Pseudomonas species constituted 50% of the isolates , Proteus
10.9% , E.coli 2.1%, Klebsiella 8.7% , and Staphylococcus 28.3% of the isolates
. Pseudomonas aeruginosa was the predominant species of Pseudomonas (56.5%)
while mixed group , Pseudomonas putida and Pseudomonus fluorscens
constitute (19.6% , 15.2% , 8.7%) respectively. All strains of Pseudomonas
were resistant to SXT , AML , SAM , CP , TE . most of the strains were
sensitive to AK , TOB , CN , CFP , PRL ( 91.3% , 89.2% , 69.6% , 56.6%) in
this descending order of activity . Most of the strains were resistant to
E(91.3%) AMC(74.9%) , CRO(63%) , ATM (56.5%) . 20 (43.4%) of strains were β-lactamase producer , out of these isolates 16 strains (80%) contained plasmid
, 4(20%) were plasmidless . Extended spectrum β- lactamase was demonstrated
in 4(8.6%) of isolates . Plasmid profile analysis showed that 18 strains
(39.1%) harbored plasmids with molecular weight ranging from 1.4 - 140 MDa
. The number of plasmid in each strain , ranged between one and five . 9
strains(50%)contained 3 plasmids while 6 (33.3%)had one plasmid, 2(11.1%
)had 2 plasmid , (5.5%) of strain contain 5 plasmids . Resistance to AK ,
TOP , ATM carried mostly on a large molecular weight plasmid . Moreover ,
resistance to increasing number of antibiotics was associated with high
molecular weight plasmids .

In conclustion the burn wound represents a site suscepitble to opportunistic
colonization , Pseudomonus aeruginosa infection is particularly problematic
, as all isolates were multidrug resistant . Most of these strains had
plasmid which may be responsible for resistance to antibiotics .
Production of ESBL is important problem in Egypt . Implementation of
infection control measures may help in reducing further transmission of
Pseudomonas clone within the Burn Units

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Search and Discovery of Antibiotics: A preliminary Data from Qatar, Gulf.
Al-Thani, R.F.
Department of Biological Sciences, Faculty of Science, University of Qatar, Doha, Qatar.

The production of a new generation of antibiotics from Streptomyces is of special importance as most pathogens have acquired antibiotic resistant capabilities. We have previously isolated from Qatar soil 34 Streptomyces isolates and identified them to species level. In this study, we screened these isolates for antimicrobial activity against known multi-antibiotic resistant (MAR) strains of bacteria (n= 16) and fungi (n=4). The general-antibiotic activity rate (GAAR) ranged from 40 to 95 % and the highest activity was shown against Staphylococcus aureus, S. epidermidis, and Escherichia coli. The specific-antibiotic activity rate (SAAR) ranged from 45-100 % and the most powerful antibiotic activity was that of the Streptomyces Q30D, Q41A and Q65D strains. These 3 strains showed a 100 % inhibition for all the tested bacterial and fungal strains and the mean value of the inhibition zones reached 21.5 mm with the range of 10-32 mm. Although most of the commercial antibiotics are ineffective against MAR pathogens, our Qatari Streptomyces antibiotic activity overcomes these problems.

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