Characterization of Enterococcus faecium isolates and first report of vanB phenotype-vanA genotype incongruence in the Middle East.

We aimed to characterize the vancomycin genotype/phenotype, carriage of putative virulence genes, and genetic relatedness of Enterococcus faecium isolates in Saudi Arabia. E. faecium isolated from inpatients at our medical center were studied. Sensitivity to ampicillin, linezolid, teicoplanin, quinupristin/dalfopristin, tetracycline, and ciprofloxacin was determined. The presence of van genes and virulence genes for aggregation substance (Asa-1), enterococcal surface proteins (esp), cytolysin (cylA, cylL, cylM), gelatinase (gelE), E. faecium endocarditis antigen (EfaA( fm )), hyaluronidase (hyl), and collagen adhesion (Ace) was assessed. Genetic relatedness was determined by pulsed-field gel electrophoresis (PFGE). Twenty-nine E. faecium isolates were obtained and the majority of isolates (n/N = 22/29) were from stool specimens. PFGE analysis identified eight pulsotypes (A-H) based on 80 % similarities. Isolates were represented in five major pulsotypes: type A (n = 5), type B (n = 3), type D (n = 6), type E (n = 5), and type F (n = 7). All isolates were vanA gene-positive. Thirteen isolates had vanA(+)/vanB(+) genotype. Of these, ten exhibited a vanB phenotype and three had a vanA phenotype. Eight isolates with vanA(+)/vanB(-) genotype exhibited vanB phenotype. Six of these eight isolates belonged to the same pulsotype. All isolates were positive for gelE, esp, and EfaA( fm ) genes. Five were CylA-positive and 24 had the hyl genes. Of the eight isolates harboring a combination of gelE, esp, EfaA( fm ), and hyl genes, five showed vanB phenotype-vanA genotype incongruence. This is the first report of vanB phenotype-vanA genotype incongruent E. faecium in the Middle East region. Molecular typing indicates clonal spread and high occurrence of virulence genes, especially esp genes, associated with epidemic clones.

Effect of the introduction of pneumococcal conjugate vaccines on serotype prevalence in Kuwait and Saudi Arabia.

BACKGROUND: Streptococcus pneumoniae infection is a major cause of morbidity and mortality. Although pneumococcal disease burden in Kuwait and Saudi Arabia is considered high, comprehensive surveillance data on pneumococcal conjugate vaccine (PCV) effects are lacking. METHODS: Sterile isolates from patients in Kuwait (2003-2016) and Saudi Arabia (aged ≤5 years, 2000-2010; all patients, 2011-2015) were included. Serotyped isolates were classified by inclusion in the 7-valent (PCV7) or 13-valent PCV (PCV13); isolates of other serotypes were classified as "non-PCV13". Isolate frequency (number of isolates/year) and classification of isolates according to vaccine type were assessed by period (before PCV, after PCV7, and after PCV13 introduction). RESULTS: In Kuwait, the frequency of collected isolates was highest after PCV7 introduction. Decreased frequency of PCV7 serotypes was seen after PCV13 introduction compared with before PCV and after PCV7 introduction. Increased frequency of the 6 additional serotypes in PCV13 and non-PCV13 serotypes was observed after PCV7 introduction with a subsequent decrease in the 6 additional serotypes in PCV13 and non-PCV13 serotypes after PCV13 introduction. The percentage of isolates of vaccine serotypes in Kuwait decreased over time. In Saudi Arabia, the frequency of collected isolates was highest after PCV7 introduction. An increased frequency of PCV7 serotypes was observed after PCV7 introduction, with a further decrease after PCV13 introduction. For the 6 additional serotypes in PCV13, an increased frequency was seen after PCV7 and PCV13 introduction compared to before PCV introduction. For non-PCV13 serotypes, an increased frequency was observed after PCV13 introduction compared to after PCV7 introduction. The percentage of isolates covered by PCV13 serotypes was similar across periods, while a substantial decrease in isolates covered by PCV7 was seen after PCV13 introduction. CONCLUSION: PCVs in Kuwait and Saudi Arabia resulted in decreased frequency of some vaccine serotypes and an emergence of some non-PCV13 serotypes. Further investigation is warranted.

Emergence and trends of penicillin non-susceptible Streptococcus pneumoniae in Saudi Arabia and Kuwait - perspective and outstanding issues.

For many years in the past Streptococcus pneumoniae was uniformly susceptible to penicillin until the sudden and unexpected emergence of clinical infections caused by penicillin-resistant S. pneumoniae (PRSP) in 1967. Within the following decade, reports of nosocomial and community outbreaks of infections due to PRSP became widespread all over the world. Recent reports suggest that the incidence of resistance rates is rising in many countries although there are geographical variations in the prevalence and patterns of resistance between countries. The problem of antibiotic resistance is further compounded by the emergence of resistance to many beta-lactam antibiotics. The first report of PRSP in Saudi Arabia was in 1991. Barely a year after, PRSP infection was reported in Kuwait in 1992. Since then, studies from various parts of these countries have recorded prevalence rates ranging from 6.2% in Riyadh to 34% in Jeddah and 20% to 56% in neighboring Kuwait. These suggest considerable variation in the prevalence of PRSP in different cities in the Saudi Kingdom and Kuwait. The mechanism of resistance is due to chromosomally mediated alteration of penicillin-binding proteins (PBPs), which are target sites for beta-lactam antibiotics. It would appear that the spread of PRSP strains in Saudi Arabia is driven by the selective pressure created by excessive use and misuse of antimicrobial agents made possible by the easy availability of these agents, often frequently obtainable over the counter. In Kuwait, irrational and misguided use of antibiotics may be the major driving force favoring the spread of PRSP. The serotypes of strains encountered in Saudi Arabia and Kuwait are almost identical, with serotypes 19, 6, 15, 14 and 23 being the most common; together they constitute about 70% of the isolates circulating in these countries. In general, almost 90% of the serotypes included in the 23-polyvalent vaccine are present in the general population. However, a much lower percentage of these serotypes is found in the conjugated vaccines, which are more relevant to our communities. This paper reviews the emergence and the steady increase in the prevalence of penicillin-resistant pneumococcal strains in Saudi Arabia and Kuwait during the last 10 years. It discusses the trends, mechanisms of resistance and factors associated with the emergence, dissemination, and colonization of resistant organisms and suggests options available to clinicians for management of infections due to PRSP.

Patterns of macrolide resistance determinants among S. pyogenes and S. pneumoniae isolates in Saudi Arabia.

In the study we characterized the macrolide sensitivity of recent clinical isolates of Streptococcus pyogenes and S. pneumoniae collected from major Saudi Arabian hospitals. Susceptibility testing was performed using standard National Committee for Clinical Laboratory Standards methodology on 335 S. pyogenes and 350 S. pneumoniae isolates. Macrolide resistance mechanism phenotypes were identified using double-disk diffusion. All S. pyogenes were penicillin sensitive, while 6.3% were macrolide resistant, the main mechanism of which was of M phenotype (96%). Approximately 51% of S. pneumoniae were penicillin non-susceptible. Macrolide resistance in S. pneumoniae accounted for 18.8%, the majority of which were M phenotype (91%). Low-level resistance mediated by mef-bearing strains pre-dominated. Newer macrolides, including azithromycin, are still considered drugs of choice for empirical treatment of respiratory infection in such circumstances.

Postantibiotic effect of roxithromycin on streptolysin O production, hydrophobicity, and bactericidal activity of PMNL by Streptococcus pyogenes.

Exposure of Streptococcus pyogenes to 5 x minimum inhibitory concentration of roxithromycin for 1 h produced a significant postantibiotic effect. More than 2.5 h was necessary for roxithromycin-treated bacteria to increase by 1 log10 in colony-forming units after drug removal, compared with the unexposed cells. After exposure to and removal of the drug, treated cells failed to exhibit normal hemolytic activity for at least 4 h. The inhibitory effect persisted for 20 h after drug removal, although the extent of growth for treated and untreated cells was almost the same. Hydrophobicity of treated cells, studied throughout the logarithmic growth phase with a water-hexadecan two-phase system, was markedly decreased by 40%, compared with untreated cells 4 h after drug removal. Cells that had been treated with roxithromycin became more susceptible to the bactericidal activity of human PMNL than untreated bacteria. The data indicate that some of the metabolic activity that contributes to the virulence of S. pyogenes is affected by postexposure to roxithromycin, and its minimum inhibitory concentration and serum level might not be the best indicators of efficacy in this class of drugs.

Effect of four antibiotics on haemolysin production and adherence to human uroepithelial cells by Escherichia coli.

The effect of subinhibitory concentrations of chloramphenicol, gentamicin, netilmicin and streptomycin in artificial culture media on growth, haemolysin production and adherence to uroepithelial cells by Escherichia coli was examined. Only streptomycin inhibited haemolysin production without inhibiting growth of the bacteria. In contrast, the inhibition of haemolysin production by chloramphenicol, gentamicin or netilmicin was directly proportional to the degree of inhibition of growth. Bacterial adherence to human uroepithelial cells, measured microscopically, was affected by all four antibiotics but to different extents. There was no correlation between inhibition of haemolysin biosynthesis and the ability of the bacteria to adhere to uroepithelial cells after growth in the presence of the antibiotics.

Antibiotic inhibition of protease production by Pseudomonas aeruginosa.

The effects of tetracycline, chloramphenicol and polymyxin B on growth and protease production by Pseudomonas aeruginosa were studied. Tetracycline inhibited protease production at concentrations much lower than those required to cause growth inhibition; the effect was not due to inhibition of protease activity by the antibiotic. In contrast, chloramphenicol and polymyxin B inhibted protease production in direct proportion to the inhibition of growth. Lysozyme-release experiments with washed tetracycline-treated cells indicated that the protease did not accumulate intracellularly. The protease-inhibiting effect of tetracycline might have therapeutic significance if it were found to occur in vivo.

Post-antibiotic effect of azithromycin and erythromycin on streptococcal susceptibility to phagocytosis.

The effect of azithromycin and erythromycin on growth, cell surface hydrophobicity and the susceptibility to the bactericidal activity of human polymorphonuclear leucocytes (PMNL) was examined in four Streptococcus species. Exposure to either 10 x MIC azithromycin or erythromycin induced a post-antibiotic effect (PAE) of between 2.4 and 4.3 h. Erythromycin caused a longer PAE for S. sanguis than azithromycin under the same conditions. The cell surface charge (hydrophobic or hydrophilic) of the streptococci was altered significantly during PAE; loss of hydrophobicity was induced by both macrolides, and this effect was variable amongst the species. The decrease in hydrophobicity was not related to inhibition of growth. The effect of each drug during PAE on the interaction of opsonised suspensions of the streptococci with human PMNL revealed that erythromycin, and to a lesser extent azithromycin, increased susceptibility to the bactericidal activity of human PMNL; this effect was abolished following PAE. The present study clearly showed that PAE should not only be considered as delayed bacterial growth, but also as modulation of bacterial susceptibility to phagocytosis which may influence the outcome of the host-parasite relationship.

Comparative efficacy of successive exposure of Pseudomonas aeruginosa to gentamicin and ceftazidime.

Aminoglycoside and beta-lactam antibiotics, when used in combination, are usually given simultaneously, however, successive administration may be more efficient. The killing capacity was used to assess the effect of time intervals between low and high concentrations (2-8xMICs) of gentamicin and/or ceftazidime on Pseudomonas aeruginosa and to determine which drug is better to be administered first. The killing capacity after exposure to the antibiotic for 1 h were compared: (i) cells treated with gentamicin alone; (ii) cells treated with ceftazidime alone; and (iii) ceftazidime was added to (i) or (iv) gentamicin was added to (ii) at 0, 1 and 3 h of antibiotic removal. The bactericidal activity of gentamicin was potentiated and the viable cells decreased up to 6 h after antibiotic removal when the ceftazidime was added at O and at 1 h but the extent of bactericidal activity was reduced, when it was added at 3 h after gentamicin removal. Alternatively, treating the cells first with ceftazidime and then gentamicin was added after drug removal at O and at 1 h resulted in a marked decline in the viable cells, while addition of gentamicin after 3 h from ceftazidime removal, the extent of bactericidal activity was reduced. The non-treated cells with gentamicin started to grow heavily within 6 h of ceftazidime removal. No viable cells were detected after overnight incubation in cultures treated first with 6 or 8xMIC of gentamicin for 0.5 or 1 h. This in vitro study suggests that the optimum interval between gentamicin and ceftazidime doses, which gave the maximum bactericidal effect and the time before re-growth, appeared to be 1-2 h.

Elucidation of antibiotic effectiveness against Staphylococcus epidermidis during intraocular lens implantation.

The effect of various antimicrobial agents commonly used in irrigating solutions on the hydrophobicity and adherence of Staphylococcus epidermidis ATCC 14990 was investigated. The longest post-antibiotic effect (PAE=3.2 h) was obtained with gentamicin followed by ciprofloxacin (2.7 h), clindamycin (2.2 h), ceftazidime (1.8 h) and vancomycin (1.6 h). The post-antibiotic effect on surface hydrophobicity of cells previously treated with gentamicin, ciprofloxacin, or clindamycin for 120 min resulted in a substantial decrease in affinities to hexadecane (31.4, 28.5 and 27%, respectively) compared with control untreated cells. Less effect was noted with cells previously treated with ceftazidime or vancomycin (20.5 and 15.8%, respectively). Similar but less marked results were obtained when the cells were exposed to antibiotics for 30 or 60 min. The post-antibiotic effect on adherence of cells to both intraocular lenses and to epithelial cells showed that adherence to lenses decreased as the time of exposure to antimicrobial agents increased. Adherence was greatly diminished with cells treated with gentamicin or ciprofloxacin compared with control untreated cells. Adherence was less affected by clindamycin, ceftazidime and vancomycin. The data supported the use of antimicrobial agents in irrigating solution during intraocular surgery, since, reduced adherence (colonization) and might result in a lower incidence of endophthalmitis.

Comparative effects of selected phenothiazine tranquilizers and antihistaminics on bacterial cells and possible interactions with antibiotics.

Evaluation of the antibacterial effect of phenothiazine antihistaminics (trimeprazine, promethazine, and fonazine) and phenothiazine tranquilizers (promazine, chlorpromazine, triflupromazine, and propiomazine) on Staphylococcus aureus showed that tranquilizers were more active [minimum inhibitory concentration (MIC) 0.5-1.6 micrograms/mL] than antihistaminics (MIC greater than 1.6 micrograms/mL). The antibacterial activity was found to correlate with both the rate of adsorption of these drugs on the bacterial cells and the surface tension of their solutions. Phenothiazine tranquilizers caused rapid and extensive leakage of potassium ions from bacterial cells, while phenothiazine antihistaminics produced relatively slower leakage of these ions. A study of the effect of the phenothiazines on the antibacterial activity of some antibiotics showed that all phenothiazines produced a synergistic effect with erythromycin and an antagonistic effect with tobramycin. Variable effects were observed with chloramphenicol, and no effect was observed with penicillin. Results were explained on the basis of structural characteristics of the phenothiazines.

Influence of various agents on adsorption capacity of kaolin for Pseudomonas aeruginosa toxin.

The in vitro adsorption of 125I-labeled Pseudomonas aeruginosa toxin (I) onto kaolin (II) at various pH values and in the presence of various agents was studied. The maximal adsorption capacity of I onto II occurred at pH values below 4.1, while minimal values were observed at pH 4.1, 7.4, and 8; average adsorption occurred at pH 5 and 6. The tested agents at specific ratios to II decreased the percent adsorption capacity of II for I to various degrees. The results are explained by reference to the structures of both kaolin and the toxin.

Influence of antibiotics on host-parasite interactions with an emphasis on in-vivo studies.

There has been considerable recent interest in the nature and importance of interactions which occur between antibiotics, microorganisms and the various cellular and humoral components of the host. The influence of antibiotics on host defense mechanisms is important because of the increasing number of compromised hosts in whom even a marginal influence on the host response may have a significant effect of the outcome. The influence of antibiotics on host/bacterial interaction reveals subtle effects on the ultrastructure of the bacterial cell, the biosynthesis of various virulence factors, the ability of microorganisms to adhere to epithelial cells and the function of polymorphonuclear leukocytes and the immune-specific response of mononuclear cells. These effects permit the proposal that at least some of the subtle effects of antibiotics on microbial pathogenicity might take place in vivo. If antibiotics can be shown definitely to enhance host-defense mechanisms, especially in compromised hosts, then more effective antibiotic therapy can be provided for these indications. Finally if antibiotics can modify the outcome of host-parasite interactions then perhaps we need to consider testing antibiotics against these parameters.

In-vitro evaluation of a new oral cephalosporin, cefroxadine (CGP 9000).

The in vitro activity of cefroxadine was studied and found to be at least comparable to that previously reported for cefalexin and cefradine. The activity of cefroxadine was superior to that of amoxicillin against tested isolates. Time-killing studies showed that the addition of 4 X minimum inhibitory concentration (MIC) of cefroxadine to growing cultures reduced viable counts by 4 log units within a 3 h incubation. A diffusion test with a 30 microgram cefroxadine disk produced acceptable interpretive results with tentative zone size breakpoints of less than or equal to 14 mm for resistance and greater than or equal to 17 mm for susceptibility.

Antibiotic resistance in developing countries.

During the past decade there have been major changes in the susceptibility of bacteria that cause various infections. Resistance to anti-infective agents, including antibiotics, is worldwide, both in developed and developing countries. Almost all bacterial species can develop resistance to anti-infective agents and resistance can readily be transferred among bacteria by transmissible elements (plasmids). Measures to prevent the emergence of resistance must be implemented urgently. A multiplicity of factors drive antibiotic resistance and solutions require the collaboration of governmental agencies, pharmaceutical companies, healthcare providers and consumers. Knowledge of resistance patterns and of the ways by which resistance is overcome is vital to the future of antimicrobial chemotherapy.

Differential inhibition by clindamycin on slime formation, adherence to teflon catheters and hemolysin production by Staphylococcus epidermidis.

Slime formation was detected in Staphylococcus epidermidis strains isolated from either infected patients or healthy individuals. Cells of S. epidermidis, that either formed slime or not, adhered to teflon catheters. There was no correlation between adherence of bacteria to teflon catheters and slime formation. Clindamycin at subinhibitory concentration significantly inhibited slime formation without inhibiting bacterial growth. Adherence of S. epidermidis to teflon catheters was affected by the presence of clindamycin whether slime was produced or not. Clindamycin at subinhibitory concentrations markedly inhibited hemolysin production by S. epidermidis without appreciably altering the cell density, and cells grown in the presence of the drug showed very low hemolytic activity upon disruption. These results suggest that clindamycin at low concentration alters S. epidermidis virulence properties, apart from inhibiting growth.

Correlation between the in vivo and in vitro antimicrobial properties of commercially available mouthwash preparations.

The effectiveness of six commercially available mouthwashes against common buccal organisms was studied. The minimum inhibitory concentrations (MIC) for two of the studied mouthwashes (Corsodyl and Oraldene) against buccal organisms were determined in Todd Hewitt medium with or without 5% serum. The concentration of the active substance in these two mouthwashes was in excess of the corresponding MIC. When the medium was supplemented with serum, lower MIC values were observed. Kill-time determinations, used at half the concentration of the normal preparation, revealed a rapid lethal effect for all tested mouthwashes. The slowest lethal effect was observed with Fluocaril mouthwash. When mouthwashes were tested in volunteers, an immediate significant fall in salivary bacterial counts was produced by all except Fluocaril. With the latter mouthwash the decrease was significant 2-30 minutes after rinsing. The bacterial levels returned to pre-rinse levels after 30 minutes for Listerine, after 90 minutes for both Oraldene and Mint and after 180 minutes for Corsodyl, Fluocaril and Sansilla mouthwashes. The results indicate that there is a good correlation between in vivo efficacy and in vitro determination of all mouthwash preparations.

Beta-lactam and macrolide resistance and serotype distribution among Streptococcus pneumoniae isolates from Saudi Arabia.

Three hundred thirty-six clinically significant Streptococcus pneumoniae isolates were collected from laboratories of different hospitals in Riyadh, Saudi Arabia. Most of these isolates were from pulmonary and otitis media (68.2%), and 31.8% were extrapulmonary (blood and CSF). Of the 336 isolates, 44.6% were susceptible to penicillin, and 55.4% were penicillin non-susceptible (35.7% were intermediate and 19.7% were fully resistant). The isolates showed 9.0% resistance to co-amoxiclav, 31.8% to cefuroxime and 39.4% to cefprozil. None of the isolates were resistant to ceftriaxone. Overall macrolide resistance rates were 22.6% to erythromycin, 18.5% to roxithromycin, 17.9% to azithromycin and 17.3% to clarithromycin. Most penicillin non-susceptible pneumococci were of serogroups/types 19 (21.0%), 6 (10.8%), 18 (8.6%), 23 (8.1%) and 14 (7.0%). Serogroups 9, 15, and 1 were found in 5.4%, 4.3%, and 2.2% of the isolates, respectively. Nontypeable strains constituted 6.5%. In exploring the mechanism of resistance to macrolides, 28 of 76 (36.8%) of isolates were erythromycin-resistant due to ribosomal mechanism (all were constitutive type, none were inducible), whereas 48 (63.2%) isolates were resistant due to an efflux mechanism. Good antibiotic control with periodical antibiotic surveillance and appropriate use of pneumococcal vaccine may improve current treatment of pneumococcal infections.

Antimicrobial activity of artemisinin and its derivatives against anaerobic bacteria.

Artemisinin and nine of its semisynthetic derivatives were tested for antibacterial activity against anaerobic, facultative anaerobic, microaerophilic and aerobic bacteria. Only anaerobic bacteria and gonococci showed sensitivity to artemisinin derivatives. Artemisinin and its deoxy derivatives had no activity at 100 micrograms/ml concentration. The newly synthesized methyl diperoxy derivative had the highest activity against all tested anaerobes with a MIC of 9.4 micrograms/ml.

Macrolides induced suppression of virulence factors produced by Staphylococcus aureus.

The effect of sub-MICs of azithromycin, clarithromycin and roxithromycin, as compared to erythromycin, on the production of coagulase, beta-hemolysin, lecithinase and deoxyribonuclease by Staphylococcus aureus was studied. All new macrolides completely inhibited coagulase and beta-hemolysin production and partially inhibited lecithinase and deoxyribonuclease. Such inhibition is not related either to growth inhibition or to inhibition of enzyme activity. Erythromycin, on the other hand, had no effect on coagulase or beta-hemolysin production but slightly suppressed the production of lecithinase and deoxyribonuclease. This inhibitory effect might have clinical significance if it was found to occur in vivo.