Edet E. Udo

Genetic analysis of methicillin-resistant Staphylococcus aureus expressing high- and low-level mupirocin resistance.

Clinical strains of methicillin-resistant Staphylococcus aureus (MRSA) expressing high- and low-level mupirocin resistance were studied to determine the genetic location of mupirocin and other resistance determinants. Mupirocin resistance was confirmed by MIC determination with E-test strips. Curing and transfer experiments were used to establish the genetic location of the resistance determinants and the PCR with mupA-specific primers was used to detect the presence of mupA genes. High-level mupirocin-resistant isolates had MICs >1024 mg/L, whereas the low-level resistant isolates had MICs of 32-128 mg/L. The isolates carried plasmids ranging from 2.8 to 38 kb in size. All of them carried 26- and 3.0-kb plasmids, but only the high-level mupirocin-resistant isolates carried a 38-kb plasmid. Curing and transfer experiments revealed that the 26-kb plasmid encoded resistance to cadmium, mercuric chloride, propamidine isethionate and ethidium bromide and the 38-kb plasmid was a conjugative plasmid encoding high-level mupirocin resistance. One isolate, IBN287, carried both plasmid-borne high-level and chromosomal low-level mupirocin resistance. The mupA gene was detected on the 38-kb plasmid DNA but not in the genomic DNA of the low-level mupirocin-resistant isolates. The genomic DNA of strain IBN287 cured of the 38-kb mupirocin resistance plasmid did not contain mupA. The results suggest that different genes encoded low- and high-level mupirocin resistance in these isolates.

Enterotoxin production by coagulase-negative staphylococci in restaurant workers from Kuwait City may be a potential cause of food poisoning

Staphylococcus aureus and coagulase-negative staphylococci (CNS) were isolated from the hands of food handlers in 50 restaurants in Kuwait City and studied for the production of staphylococcal enterotoxins, toxic shock syndrome toxin-1, slime and resistance to antimicrobial agents. One or a combination of staphylococcal enterotoxins A, B or C were produced by 6% of the isolates, with the majority producing enterotoxin B. Toxic shock syndrome toxin-1 was detected in c. 7% of the isolates; 47% produced slime. In all, 21% of the isolates were resistant to tetracycline and 11.2% were resistant to propamidine isethionate and mercuric chloride. There was no correlation between slime and toxin production or between slime production and antibiotic resistance. The detection of enterotoxigenic CNS on food handlers suggests that such strains may contribute to food poisoning if food is contaminated by them and held in conditions that allow their growth and elaboration of the enterotoxins. It is recommended that enterotoxigenic CNS should not be ignored when investigating suspected cases of staphylococcal food poisoning.

Rapid detection of methicillin resistance in staphylococci using a slide latex agglutination kit

The slide latex agglutination test, MRSA-Screen, was compared with the mecA polymerase chain reaction (PCR) and traditional susceptibility test methods for the detection of methicillin resistance in Staphylococcus aureus and coagulase-negative staphylococci. The MRSA-Screen test detected the same number of methicillin-resistant S. aureus as the mecA PCR and the traditional susceptibility tests. It correctly identified all 21 methicillin-susceptible S. aureus as being sensitive. It also produced the same result as the mecA PCR in identifying a methicillin-resistant S. aureus among six isolates classified as borderline resistant by traditional susceptibility tests. The MRSA-Screen test and mecA PCR detected methicillin resistance in 10 and 15 of 17 methicillin-resistant coagulase-negative staphylococci, respectively. From these results, it is concluded that the MRSA-Screen is a very accurate, reliable and rapid method of detecting methicillin resistance in S. aureus and is suitable for use in clinical microbiology laboratories. Further study of its use in detecting methicillin resistance in coagulase-negative staphylococci is required.

Synthesis and antibacterial activity of 5-substituted oxazolidinones.

A series of 5-substituted oxazolidinones with varying substitution at the 5-position of the oxazolidinone ring were synthesized and their in vitro antibacterial activity was evaluated. The compounds demonstrated potent to weak antibacterial activity. A novel compound (PH-027) demonstrated potent antibacterial activity, which is comparable to or better than those of linezolid and vancomycin against antibiotic-susceptible standard and clinically isolated resistant strains of gram-positive bacteria. Although the presence of the C-5-acetamidomethyl functionality at the C-5 position of the oxazolidinones has been widely claimed and reported as a structural requirement for optimal antimicrobial activity in the oxazolidinone class of compounds, our results from this work identified the C-5 triazole substitution as a new structural alternative for potent antibacterial activity in the oxazolidinone class.

Synthesis and antibacterial activity of novel 5-(4-methyl-1H-1,2,3-triazole) methyl oxazolidinones.

A series of 5-(4-methyl-1,2,3-triazole)methyl oxazolidinones were synthesized and tested for their antibacterial activity against a panel of Gram-positive and Gram-negative clinical isolates in comparison with linezolid and vancomycin. Most of the compounds demonstrated strong to moderate in vitro antibacterial activity against susceptible and resistant Gram-positive pathogenic bacteria. Antibacterial activity varied with substitutions at the phenyl C4 position with bulky alkylcarbonyl and alkoxycarbonyl substitutions on the piperazine N4 being detrimental to antibacterial activity. Whereas the presence of the 4-methyl-1,2,3-triazole moiety in the acyl-piperazine containing analogs resulted in increased protein binding, and decreased antibacterial activity particularly against Streptococcus pneumoniae strains.

Etiology and Antibiotic Susceptibility Patterns of Community-Acquired Urinary Tract Infections in a Kuwait Hospital

Objective: The aim of this study was to determine the distribution and antibiotic susceptibility patterns of bacterial strains isolated from patients with community-acquired urinary tract infections (UTIs) at the Infectious Diseases Hospital, Kuwait. Materials and Methods: The study was conducted over a 7-year period. Patient information was obtained from medical record files. Antibiotic-sensitivity testing was performed by disk diffusion. E test and double disk diffusion methods were used to study the production of extended spectrum β-lactamases. Results: Of the 14,042 urine samples processed, significant bacteriuria (>105 cfu/ml) was detected in 1,606 (11.4%). The majority (74.5%) of the isolates were from women while the remaining 25.5% were from men. The majority of infections (75%) were due to Enterobacteriaceae, coagulase-negative staphylococci (10.3%) and group B streptococci (8.7%). Among the gram-negative enteric bacilli high prevalence of resistance to ampicillin, amoxicillin/clavulanic acid, cephalothin, and trimethoprim/sulfamethoxazole was observed. Increasing resistance to ciprofloxacin and gentamicin was observed in E. coli isolates over the 7 years. Multiple resistance was detected in 53.8 and 41% of E. coli and Klebsiella spp. strains, respectively. No glycopeptide-resistant enterococci were isolated. Conclusion: This study revealed that Enterobacteriaceae were the predominant bacterial pathogen of community-acquired UTIs in Infectious Diseases Hospital, Kuwait. It also demonstrated an increasing resistance to ciprofloxacin, gentamicin and the production of extended spectrum β-lactamase among UTI pathogens in the community.

Origin and Evolution of European Community-Acquired Methicillin-Resistant Staphylococcus aureus

ABSTRACT Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) was recognized in Europe and worldwide in the late 1990s. Within a decade, several genetically and geographically distinct CA-MRSA lineages carrying the small SCCmec type IV and V genetic elements and the Panton-Valentine leukocidin (PVL) emerged around the world. In Europe, the predominant CA-MRSA strain belongs to clonal complex 80 (CC80) and is resistant to kanamycin/amikacin and fusidic acid. CC80 was first reported in 1993 but was relatively rare until the late 1990s. It has since been identified throughout North Africa, the Middle East, and Europe, with recent sporadic reports in sub-Saharan Africa. While strongly associated with skin and soft tissue infections, it is rarely found among asymptomatic carriers. Methicillin-sensitive S. aureus (MSSA) CC80 strains are extremely rare except in sub-Saharan Africa. In the current study, we applied whole-genome sequencing to a global collection of both MSSA and MRSA CC80 isolates. Phylogenetic analyses strongly suggest that the European epidemic CA-MRSA lineage is derived from a PVL-positive MSSA ancestor from sub-Saharan Africa. Moreover, the tree topology suggests a single acquisition of both the SCCmec element and a plasmid encoding the fusidic acid resistance determinant. Four canonical SNPs distinguish the derived CA-MRSA lineage and include a nonsynonymous mutation in accessory gene regulator C (agrC). These changes were associated with a star-like expansion into Europe, the Middle East, and North Africa in the early 1990s, including multiple cases of cross-continent imports likely driven by human migrations. IMPORTANCE With increasing levels of CA-MRSA reported from most parts of the Western world, there is a great interest in understanding the origin and factors associated with the emergence of these epidemic lineages. To trace the origin, evolution, and dissemination pattern of the European CA-MRSA clone (CC80), we sequenced a global collection of strains of the S. aureus CC80 lineage. Our study determined that a single descendant of a PVL-positive methicillin-sensitive ancestor circulating in sub-Saharan Africa rose to become the dominant CA-MRSA clone in Europe, the Middle East, and North Africa. In the transition from a methicillin-susceptible lineage to a successful CA-MRSA clone, it simultaneously became resistant to fusidic acid, a widely used antibiotic for skin and soft tissue infections, thus demonstrating the importance of antibiotic selection in the success of this clone. This finding furthermore highlights the significance of horizontal gene acquisitions and underscores the combined importance of these factors for the success of CA-MRSA. With increasing levels of CA-MRSA reported from most parts of the Western world, there is a great interest in understanding the origin and factors associated with the emergence of these epidemic lineages. To trace the origin, evolution, and dissemination pattern of the European CA-MRSA clone (CC80), we sequenced a global collection of strains of the S. aureus CC80 lineage. Our study determined that a single descendant of a PVL-positive methicillin-sensitive ancestor circulating in sub-Saharan Africa rose to become the dominant CA-MRSA clone in Europe, the Middle East, and North Africa. In the transition from a methicillin-susceptible lineage to a successful CA-MRSA clone, it simultaneously became resistant to fusidic acid, a widely used antibiotic for skin and soft tissue infections, thus demonstrating the importance of antibiotic selection in the success of this clone. This finding furthermore highlights the significance of horizontal gene acquisitions and underscores the combined importance of these factors for the success of CA-MRSA.

Genetic Lineages of Community-Associated Methicillin-Resistant Staphylococcus aureus in Kuwait Hospitals

ABSTRACT Twenty-six community-associated methicillin-resistant Staphylococcus aureus (CAMSRA) isolates were characterized by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) and screened for accessory gene regulator (agr), capsular polysaccharide (cap), and Panton-Valentine leucocidin (PVL) genes. They exhibited five PFGE patterns (types A to E). The majority were PFGE type A (12 isolates) or type B (8 isolates). MLST showed that PFGE type A isolates belonged to sequence type 80 (ST80), while the PFGE type B isolates were ST30. The ST80 and ST30 clones contained agr allotype 3, cap type 8, and PVL. The results showed that two internationally recognized CAMRSA clones are dominant in Kuwait hospitals.

Contour-clamped homogeneous electric field electrophoresis of Staphylococcus aureus

Contour-clamped homogeneous electric field (CHEF) electrophoresis is a technique of pulsed-field gel electrophoresis that enables the resolution of large fragments of DNA that cannot be resolved by conventional gel electrophoresis. The procedure involves the application of controlled electric fields that change direction at a predetermined angle to samples of DNA that have been embedded in an agarose gel matrix and digested with a restriction endonuclease. Adjustment of the electrophoresis conditions enables the separation of DNA fragments with lengths from 10 kilobases up to 9 megabases in a size-dependent manner in agarose gels. The banding patterns can be used for epidemiological typing, the separated DNA can be immobilized onto a membrane and used for genetic mapping, or individual fragments can be extracted and used for downstream genetic manipulations. The protocol requires specialized equipment and can be completed in a maximum of 7 days.

The prevalence of antimicrobial resistance and carriage of virulence genes in Staphylococcus aureus isolated from food handlers in Kuwait City restaurants

BackgroundStaphylococcus aureus is a major cause of food poisoning due to their ability to produce enterotoxins which if ingested in sufficient amounts results in sickness. Food handlers carrying enterotoxin-producing S. aureus in their noses or hands can contaminate food leading to food poisoning. We characterized 200 S. aureus obtained from food handlers in different restaurants for antibacterial resistance and the carriage of virulence genes.FindingsSusceptibility to antibacterial agents was determined by disk diffusion and Etest. PCR was used to detect genes for accessory gene regulator (agr); capsular polysaccharide (cap) 5 and 8, staphylococcal enterotoxins (SE), toxic shock syndrome toxin-1 (TSST-1) and Panton-Valentine leukocidin (PVL). Isolates were typed using pulsed-field gel electrophoresis. In total 185 (92.5%) of the 200 isolates expressed resistance to antibacterial agents. They were resistant to penicillin G (82.0%), tetracycline (19.0%), erythromycin (2.5%), clindamycin (2.0%), trimethoprim (7.5%), kanamycin (2.5%), streptomycin (1.5%), ciprofloxacin (1.5%), fusidic acid (1.0%) and cadmium acetate (68.0%). Seventy-six (38.0%) and 114 (57.0%) isolates had type 5 and type 8 capsular polysaccharides respectively. The agr types I, II and III alleles were detected in 50.5%, 20.0% and 23.5% of the isolates respectively. They contained genes for SEI (38.5%), SEG (24.0%), SEC (23.0%), SEB (12.5%), SEH (21.5%), SEA (11.0), SED (1.5%), SEE (1.5%), TSST-1 (4.0%) and PVL (9.0%).ConclusionThis study revealed a high prevalence of antibacterial resistance and virulence determinants in S. aureus from food handlers in Kuwait restaurants justifying the screening of food handlers to detect and treat carriers and protect restaurant customers from staphylococcal food poisoning.