Antagonistic interactions between the flavonoids hesperetin and naringenin and beta-lactam antibiotics against Staphylococcus aureus.

Flavonoids are a group of polyphenolic plant compounds with a range of biological activities. This study shows that the flavonoids hesperetin and naringenin have antibacterial activity against methicillin-sensitive and methicillin-resistant Staphylococcus aureus isolates. Minimum inhibitory concentrations for hesperetin were 250 and 500 microg/mL, respectively, and for naringenin were 125 and 250 microg/mL, respectively. This effect was reversed by the beta-lactam antibiotics methicillin, penicillin and oxacillin, but not by cefoxitin. For bacteria growing in the presence of these antibiotics, the flavonoids had no effect on the levels of beta-lactamase enzymes and PBP-2' compared to controls. Electron microscopy showed abnormal morphology in bacteria treated with subinhibitory concentrations of flavonoids. These results are interesting because previous studies have reported synergistic interactions between flavonoids and beta-lactam antibiotics. It is suggested that an interaction removes both inhibitors from the bacterial growth milieu.

Salmonella enterica Serotype typhi in Kuwait and its reduced susceptibility to ciprofloxacin.

Salmonella enterica serotype typhi continues to be an important public health problem in Kuwait. Analysis of the isolates from 163 patients, collected between 1995 and 2003, showed that the majority were from patients from the Indian sub-continent, including 45 from Bangladesh, 38 from India and 30 from Pakistan. Fifty-four of the strains showed multiple antibiotic resistance (MDR). Twenty-five strains were from Kuwaitis, with 15 aged <18 years. Bacteriophage typing of 20 isolates from Kuwaitis revealed that they belonged to 8 different phage types, and that the 3 MDR strains were phage type E1. Random amplified polymorphic DNA typing showed genetic variability amongst isolates from Kuwaiti patients. This method conveniently demonstrated the identity of 4 isolates associated with a small outbreak. 48 isolates from 2002-3 were tested for reduced susceptibility to quinolones. 12 of 18 MDR strains and 7/30 susceptible strains showed reduced susceptibility to ciprofloxacin (minimum inhibitory concentration 0.125-0.5 mg/L). All 12 strains were tested for mutation in the quinolone resistance determining region (QRDR) of the gyr A gene. The mutation ser83 phe was detected in the 10 strains tested. Thus typhoid fever in Kuwait is predominantly associated with those who have traveled from endemic areas to work in Kuwait. The incidence of MDR strains remains at about 30%. Reduced susceptibility to ciprofloxacin in MDR S. typhi has increased from (11%) in 1995-1996 to (67%) in 2002-2003 and from (0%) to (23%) in susceptible strains. Mutation of the gyrA gene is the mechanism most often responsible.

Extended-spectrum beta-lactamase-producing Escherichia coli isolated in the Al-Amiri Hospital in 2003 and compared with isolates from the Farwania hospital outbreak in 1994-96 in Kuwait.

Extended-spectrum beta-lactamases (ESBLs) are a major problem in Kuwait and an accurate method for their detection is essential. This study was designed to evaluate the efficacy of the commercial system (Vitek 2) to identify ESBLs in clinical isolates of Escherichia coli and relate this to their identification by agar dilution methods for use in a diagnostic laboratory. The presence of the major ESBLs parental enzyme groups was confirmed by PCR and the similarity of the strains was determined by pulsed field gel electrophoresis (PFGE) on DNA, cleaved using XbaI endonuclease, to identify clonal spread.Seventy-one separate E. coli isolates from 65 patients were tested. Sixty-two isolates were from 56 patients from the Al-Amiri Hospital and nine isolates from neonates from Farwania Hospital. The isolates were screened for ESBL activity by the Vitek 2 system. Isolates showing positive results were further tested with Etest ESBL strips and by the disc approximation methods. All the isolates were flagged as ESBL-positive by the Vitek 2 advanced expert system (AES). Isolates from all the 65 patients were detected as ESBL positive by the Etest, only if both ESBL strips were used. The double disc approximation test using five different antibiotics could detect ESBL presence in isolates from only 46 patients. In this test, the synergy with cefepime was the most sensitive in ESBL detection, showing their presence in 41 isolates. PCR with primers for bla(TEM) and bla(SHV) demonstrated that one or both of these enzymes in all isolates. PFGE revealed that many different clones were present amongst the isolates. The epidemiology of ESBL E. coli in Kuwait is complex. Many distinct strains are already present in the population, as shown by the results of PFGE. Several testing methods may be required to detect all strains harboring ESBLs.

Antibacterial resistance and molecular typing of methicillin-resistant Staphylococcus aureus in a Kuwaiti general hospital.

OBJECTIVE: To investigate antibiotic resistance and genetic relatedness of methicillin-resistant Staphylococcus aureus (MRSA) isolated in a general hospital in Kuwait over a period from 1996 to 1998 and 2001. MATERIAL AND METHODS: The isolates were characterized by antibacterial susceptibility testing, coagulase serotyping, coagulase gene polymorphism (coag-RFLP) and pulsed-field gel electrophoresis (PFGE). RESULTS: The MRSA isolates were highly resistant to gentamicin, kanamycin, ciprofloxacin, tetracycline, fusidic acid and mupirocin. The prevalence of gentamicin, kanamycin, streptomycin, tetracycline and erythromycin resistance remained high (80-96%) throughout the study period, but the prevalence of resistance to ciprofloxacin, fusidic acid and mupirocin steadily increased. The already high mupirocin resistance level increased from 12.5 in 1996, to 85.7% in 2001, and the fusidic acid resistance varied between 70.8 and 85.7%. In contrast, chloramphenicol and trimethoprim resistance declined from 25 and 29% in 1996 to 4.7 and 14.2% in 2001, respectively. The majority (91.5%) of the isolates were coagulase serotype 4. AluI restriction endonuclease analysis of amplified coagulase gene generated four coag-RFLP patterns: 92% of them were coag-RFLP type 1, while types 2, 3 and 4 were 3.5, 4.6 and 1.1% respectively. PFGE differentiated them into seven pulsotypes (PFGE types 1-7). The PFGE type 1 pulsotype constituted 90.2% of the isolates. Isolates with the type A coag-RFLP also had the type1 PFGE pulsotypes. CONCLUSION: The concordant results of PFGE and coag-RFLP demonstrated the presence of a persistent MRSA clone in the hospital during the study period.

Antimicrobial activity of a series of 1-alkyl-2-(4-pyridyl)pyridinium bromides against Gram-Positive and Gram-Negative bacteria.

OBJECTIVE: To test a series of 1-alkyl-2-(4-pyridyl)pyridinium bromides with alkyl chains containing between 9 and 16 carbons against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli, Stenotrophomonas maltophilia, Acinetobacter baumannii and Pseudomonas aeruginosa) bacteria. MATERIALS AND METHODS: Chemical synthesis was based on the reaction of 2,4'-bipyridyl with alkyl bromide. Antimicrobial activity of the bipyridyls was measured by growing bacterial cultures on Mueller-Hinton agar in the presence and absence of inhibitors. RESULTS: The compounds were most active against S. aureus. The most active compounds had alkyl chain lengths of between 11 and 16 carbons. Methicillin-sensitive S. aureus was more susceptible to the inhibitors than methicillin-resistant S. aureus (MRSA). Two subclasses of MRSA existed which differed in their susceptibility to the inhibitors. The susceptibility of MRSA strains to the compounds was increased in the presence of the efflux pump inhibitor reserpine. The activity of the compounds against Gram-negative organisms was increased when the membrane-permeabilizing agent sodium citrate was introduced. Critical micelle concentrations of the compounds were much higher than minimum inhibitory concentrations of the inhibitors. CONCLUSION: The mechanism of action of the compounds may involve perturbing bacterial membranes. The resistance of some MRSA strains to the compounds may be related to efflux pumps.

Antimicrobial susceptibility, phage typing and plasmid profile of Salmonella enterica serotype paratyphi A strains isolated in Kuwait.

OBJECTIVE: To determine the antimicrobial susceptibility, phage type and plasmid profile pattern of Salmonella enterica serotype paratyphi A strains isolated in Kuwait. MATERIAL AND METHODS: From January 1995 to December 1999, 106 strains of S. enterica serotype paratyphi A isolated from an equal number of cases of enteric fever, attending the Infectious Disease and Mubarak Al-Kabeer Hospitals in Kuwait were investigated. The isolates were tested for antimicrobial susceptibility to 8 commonly used antimicrobial agents. Their phage type and plasmid profile patterns were determined using an international set of phages and Qiagen plasmid mini kit, respectively. RESULTS: All of the isolates were susceptible to ciprofloxacin, cefuroxime, ceftazidime, piperacillin and co-trimoxazole. One hundred isolates were susceptible to ampicillin, 99 to chloramphenicol and 98 to tetracycline. None of the isolates was multidrug resistant. Sixty-six percent of the isolates were phage type I, 27.4% phage type II and 6.6% were untypable. All phage type I and untypable strains had 3 plasmids of 2.2, 5 and 20 kb, whereas phage type II strains had only 1 plasmid of 20 kb. CONCLUSION: The findings indicate that while all of the isolates of the S. enterica serotype paratyphi A were susceptible to 4 of the drugs tested, some were resistant to ampicillin, chloramphenicol or tetracycline, thereby indicating the need for continued surveillance and monitoring of antimicrobial susceptibility of these isolates.

Reduced visual acuity impairs place but not cued learning in the Morris water task.

The Morris water task is a standard method for testing spatial learning in rodents. In a place version of the task, animals utilize multiple visual cues to learn the location of a hidden platform. The ability of animals to locate a cued platform is often used to qualitatively test for possible non-cognitive contributions to deficient place learning, including reduced visual function. We investigated the role of visual acuity in water maze performance quantitatively by depriving rats of pattern vision during a critical period for visual plasticity, which reduced their acuity by approximately 27% and then tested them in typical place and cued platform configurations of the Morris water task. Animals with reduced visual acuity had a significant deficit in place learning, but eventually reached the same escape latency as non-deprived animals. Deprived and non-deprived animals, however, did not differ in their ability to locate a cued platform following place learning. These data indicate that reduced visual acuity in rats can influence measurement of their place learning and that a typical cued platform version of the task cannot detect a modest, but significant, visual deficit.

Extended-spectrum beta-lactamase-producing Klebsiella spp.

Extended-spectrum beta-lactamases (ESBLs) are associated particularly with Klebsiella spp. These enzymes have arisen by mutation of the genes coding for clavulanate-sensitive, plasmid-mediated beta-lactamases such as TEM-1, TEM-2 and SHV-1. Amino acid changes in ESBLs confer enhanced hydrolysis of oxyimino-aminothiazolyl beta-lactams and aztreonam. Enzyme hyperproduction and loss of porins contribute to hydrolytic efficiency. ESBLs are highly susceptible to inhibition by clavulanate, and their presence can be detected by the disc-approximation test, using amoxycillin/clavulanate and an ESBL-susceptible antibiotic. Other manual procedures have been used and commercial tests to detect the enzymes include Etest, Vitek and Dade Microscan products. The epidemiology of ESBLs is complex, and epidemic and sporadic strains may be encountered in the same hospital. Spread between hospitals--even countries--has been documented. ESBL activity is carried on large plasmids that often carry determinants for resistance to aminoglycosides and other antibiotics, and this is transmissible to Escherichia coli and other species of Enterobacteriaceae in which ESBLs have been detected.

Experience-dependent plasticity of visual acuity in rats.

Rats have become a popular model for investigating the mechanisms underlying ocular dominance plasticity; however, no quantitative assessment of the effects of visual deprivation on behavioural acuity has been reported in this species. We measured the spatial acuity of monocularly and binocularly deprived rats with a visual discrimination task. The average spatial acuity of normal rats and rats deprived of vision after postnatal day 40 was approximately 1 cycle/degree. Monocular deprivation up to postnatal day 40 resulted in a 30% decrease in acuity and there was no recovery after 8 months. Identical binocular deprivation produced a comparable but significantly smaller reduction in acuity. The deleterious effects of monocular and binocular deprivation on visual acuity indicate that the development of cortical receptive field properties related to spatial tuning are affected by both monocular and binocular deprivation. The similarities in the effects of visual deprivation on visual acuity between rats and other mammals confirm that rats are a good model system for studying the cellular and molecular mechanisms underlying experience-dependent visual plasticity.

Behavioral assessment of visual acuity in mice and rats.

We have developed a simple computer-based discrimination task that enables the quick determination of visual acuities in rodents. A grating is displayed randomly on one of two monitors at the wide end of a trapezoidal-shaped tank containing shallow water. Animals are trained to swim toward the screens, and at a fixed distance, choose the screen displaying the grating and escape to a submerged platform hidden below it. Both mice and rats learn the task quickly. Performance falls below 70% when the spatial frequency is increased beyond 0.5 cycles in most C57BU6 mice, and around 1.0 cycles per degree (cpd) in Long-Evans rats.

Speciation of presumptive viridans streptococci from early onset neonatal sepsis.

Twenty isolates resembling viridans streptococci, 16 from blood and four from gastric aspirates, from 17 cases of early onset neonatal sepsis were identified by the API20 Strep, Rapid ID 32 Strep and conventional tests plus hydrolysis of methylumbelliferyl glycoside substrates. Nineteen of the isolates were identified as species of viridans streptococci and one as a Leuconostoc sp. Ten of the isolates were Streptococcus oralis, three S. mitis biotype 1, two S. mitis biotype 2 and one each of S. sanguis, S. vestibularis, S. salivarius and S. intermedius. The Rapid ID 32 Strep and conventional plus methylumbelliferyl tests gave the same species identity for 17 of the isolates. S. intermedius was identified by the Rapid ID 32 Strep as S. constellatus and S. salivarius as S. equinus, with S. salivarius at lower probability. The API20 Strep failed to identify S. vestibularis and identified S. salivarius as S. defectivus. The absence of certain critical tests, including urea hydrolysis, does not allow the API20 Strep to identify all the currently recognised species of viridans steptococci. The species distribution was unexpected and the incidence of S. oralis and other viridans streptococci in vaginal swabs from prenatal patients is being investigated further.

Comparison of five methods for the determination of arginine hydrolysis by viridans streptococci.

Seventy-eight clinical isolates and four control strains of viridans streptococci were tested in parallel for arginine hydrolysis by five different methods. These comprised two commercial systems, the API20 STREP and Vitek GPI card, two published methods, one based on ammonia production and one on alkalisation of Møeller's decarboxylase medium, and a method based on alkalisation of a phenol-red broth medium dispensed into microtitration plates. The clinical isolates were speciated by their biochemical reactions in the API20 STREP and API20 ZYM systems. One strain produced only a weak reaction for arginine hydrolysis in the medium by ammonia production, but otherwise the results with this medium, the API20 STREP and the microtitration plate method were identical. Tests with the Møeller decarboxylase medium and Vitek GPI card gave negative results with isolates that were positive by other methods. Inoculum size was shown to influence arginine hydrolysis obtained with Streptococcus sanguis NCTC 7863 and S. milleri 10713.

Plasmid-mediated multidrug resistance in Salmonella typhi in Kuwait.

Multidrug resistant Salmonella typhi infection is common in Kuwait. Between January 1993 and December 1994, 266 strains of S. typhi were isolated from blood of suspected typhoid fever cases attending the Infectious Disease Hospital, Kuwait. Of these, 205 strains were isolated from patients from the Indian sub-continent, 105 (45%) of which showed resistance to one or more drugs; 91 of these resistant strains showed resistance to the oral antimicrobials ampicillin, chloramphenicol, tetracycline and co-trimoxazole (ACTCo). All 266 isolates were sensitive to fluoroquinolones and cephalosporines. The minimum inhibitory concentrations (MIC) for ampicillin, chloramphenicol and tetracycline in resistant strains were > 1000 micrograms/ml. All 91 strains with ACTCo resistance pattern transferred their resistance to a recipient E. coli strain, whereas none of the remaining 14 strains with different resistance patterns transferred theirs. This paper reaffirms that multidrug resistant S. typhi is quite common in Kuwait and that such resistance is encoded by auto-transferring plasmids.

Childhood shigellosis: clinical and bacteriological study.

Ninety-three children with shigellosis were hospitalized in a regional hospital in Kuwait during the year 1988. S. flexneri was the most common isolate (54%) followed by S. sonnei (39%) and S. boydii (7%). In addition to the gastrointestinal manifestations, 14 patients (15%) developed generalized convulsions. The benign and self-limiting nature of the convulsions associated with shigellosis means that neither diagnostic procedure nor drug therapy are usually necessary. The disease was self-limiting in 41% and antimicrobial usage did not seem to shorten the duration of symptoms or hospital stay. All shigellae isolated were sensitive to gentamicin and amikacin, 56% were resistant to three or more antimicrobial agents, 32 were resistant to five or more. These findings may indicate the need for better control of antibiotic use, particularly in developing countries.

Childhood bacterial meningitis in Kuwait.

A total of 110 cases of bacterial meningitis were studied over a 7-year period. The attack rate was 3.2/100,000 overall and 13/100,000 in children under 12 years. Haemophilus influenzae was the most common pathogen (44.3%), followed by Streptococcus pneumoniae (20.9%) and Neisseria meningitidis (12.7%). The mean patient age was 2.7 years, but 44/49 cases of H. influenzae meningitis were in children under 2 years, an attack rate of 31/100,000. The mortality was 22.4% for neonates and 6.5% for other children, and was associated with coma at presentation and unusual microorganisms. Neurological sequelae were detected in 19% of surviving children.

Competitive processes in a monoculture of Pinus radiata D. Don.

The changes between 9 and 40 years of age of the frequency distribution of tree diameters were studied in an experiment in a plantation of Pinus radiata D. Don in South Australia in which the spacing between trees at planting varied. Empirical functions were developed to relate tree diameter to growth rate in diameter, variance of growth rate in diameter and mortality rate. These functions were used in solving a forward diffusion equation to predict future diameter distributions. The contributions from each of the terms in the diffusion equation were examined and it was found that the drift (growth rate in diameter) term made the greatest contribution to the solution, whilst the diffusion (variance in growth rate) term had neglible effect. This implied that competition between individual trees for light was the dominant competitive process operating in this plantation, rather than below ground competition for soil resources. The ramifications of this finding to forest growth modelling are discussed.

Evaluation of a latex kit reagent for the detection and identification of pneumococci.

The ability of a commercial latex kit to detect pneumococcal antigen in 50 CSF samples, 150 ear swabs and 80 sputum samples was investigated. Results were compared with microscopic and culture findings. 18/19 culture-positive specimens were latex-positive. 7 latex-positive specimens yielded organisms other than S. pneumoniae which agglutinated the latex, 4 Klebsiella spp, 2 Staphylococcus aureus and 1 Haemophilus influenzae. For 11 other specimens latex agglutination was positive and culture negative. S. pneumoniae was recovered from the blood of 1 of these patients and, in 2 further cases, microscopy showed poorly stained organisms which had some resemblance to S. pneumoniae. For 10 different strains of S. pneumoniae suspensions containing between 10(4) and 10(7) organisms per ml were required to agglutinate the latex.