Characterization of high-level aminoglycoside-resistant enterococci in Kuwait hospitals.

This study investigated the distribution of genes for aminoglycoside-modifying enzymes (AME) and the genetic relatedness of high-level aminoglycoside-resistant enterococci isolated in Kuwait hospitals. A total of 117 enterococci, consisting of 109 Enterococcus faecalis, seven Enterococcus faecium, and one Enterococcus casseliflavus were studied. The MICs of gentamicin, kanamycin, amikacin, tobramycin, and streptomycin were determined by agar dilution and the genes encoding the AAC(6')- APH(2"), ANT(4'), APH(3'), APH (2")-Ib, APH (2")-Ic, APH (2")-Id, and ANT(6) enzymes were amplified by PCR. They were typed by pulsed-field gel electrophoresis (PFGE). Filter mating was used to transfer gentamicin resistance determinants. They were all resistant to kanamycin (MIC 2000 mg/L). Fifty-five isolates were resistant to gentamicin (MIC 500 mg/L), 72 were resistant to tobramycin (MIC 64 mg/L), 115 were resistant to amikacin (MIC 64 mg/L), and 97 were resistant to streptomycin (MIC 1000 mg/L). The aac(6')-Ie-aph(2")-Ia was detected in all isolates with gentamicin MIC 500 mg/L and in 15 isolates with gentamicin MIC 256 mg/L. The aph(3')-IIIa gene was detected in 101 isolates, whereas the ant(6')-Ia gene was detected in 85 of the 97 streptomycin-resistant isolates with MIC 1000 mg/L. The aac(6')-Ii gene was detected only in the seven E. faecium isolates. None of them contained ant(4')-Ia, aph(2")-Ib, aph(2")-Ic and aph(2")-Id. PFGE revealed heterogeneous patterns with no dominant clone. The results demonstrated that AME are common in aminoglycoside-resistant enterococci isolated in Kuwait. However, the absence of a dominant clone suggests that they acquired high-level aminoglycoside independently.

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.

The spread of a mupirocin-resistant/methicillin-resistant Staphylococcus aureus clone in Kuwait hospitals.

High-level mupirocin- and methicillin-resistant Staphylococcus aureus (MRSA) isolated from five hospitals in Kuwait were studied by pulsed-field gel electrophoresis (PFGE) to determine their relatedness to one another and to high-level mupirocin-resistant MRSA isolated previously in a Burns Unit. The genetic location of mupirocin resistance determinant was also determined. All of the isolates were resistant to gentamicin, kanamycin, streptomycin, tetracycline and cadmium, and contained plasmids of 38, 26 and 2.8 kb. Two isolates contained additional 4.4-kb plasmids. Transfer experiments demonstrated that the 38-kb plasmid encoded high-level mupirocin resistance and the 4.4-kb plasmid encoded chloramphenicol resistance. PFGE typing of representative isolates from the five hospitals demonstrated that the majority of them had identical or closely related pulsed-field patterns suggesting that they had a common origin. However, they differed from high-level mupirocin-resistant MRSA isolated previously in the Burns Unit in their resistance and pulsed-field patterns. Whereas the majority of the previous isolates were susceptible to ciprofloxacin and resistant to trimethoprim and chloramphenicol, the majority of the current isolates were susceptible to trimethoprim and chloramphenicol, and resistant to ciprofloxacin. Only one of the current isolates had identical pulsed-field pattern to the majority of isolates obtained previously in the Burns Unit. The results suggested that a previously dominant clone of high-level mupirocin-resistant MRSA has been replaced in the Burns Unit by a new clone, which also spread in four other hospitals.

A cadmium resistance plasmid, pXU5, in Staphylococcus aureus, strain ATCC25923.

A 25.9-kb plasmid, pXU5, encoding high level cadmium resistance was isolated from Staphylococcus aureus strain ATCC25923. A labelled cadA probe from plasmid pI258 hybridised to a 2.3-kb EcoRI fragment of pXU5. pXU5 was incompatible with an S. aureus incompatibility group 1 plasmid.

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.

Characterisation of methicillin-resistant Staphylococcus aureus from Kuwait hospitals with high-level fusidic acid resistance.

Forty-seven fusidic acid- and methicillin-resistant Staphylococcus aureus isolates from clinical samples in four hospitals in Kuwait were studied for their relatedness by biotyping and pulsed-field gel electrophoresis (PFGE) and for the genetic location of their resistance determinants. Forty-four isolates were resistant to gentamicin, kanamycin and neomycin. Forty-one isolates were resistant to erythromycin and trimethoprim, 10 were resistant to chloramphenicol and four were resistant to ciprofloxacin. They contained two or three plasmids of c. 28, 2.8 and 1.8 kb. Genetic studies demonstrated that resistance to cadmium, propamidine isethionate and ethidium bromide were linked and were carried on the c. 28-kb plasmid. Chloramphenicol resistance was encoded by the 2.8-kb plasmid in resistant isolates. No resistance was associated with the 1.8-kb plasmid and this was considered to be a cryptic plasmid. Resistance to fusidic acid, methicillin, benzylpenicillin, gentamicin, kanamycin, neomycin, tetracycline, trimethoprim, erythromycin and ciprofloxacin were located on the chromosome. All the isolates produced urease, but varied in the production of haemolysins, pigments, lipase and lecithinase and were classified into nine biotypes. In contrast, PFGE divided the isolates into two major patterns with one PFGE type constituting the majority of isolates in all four hospitals. The presence of the dominant PFGE pattern in all four hospitals suggests that it is an epidemic MRSA clone with the capacity to spread. Infection control measures should be directed towards restricting the further spread of this clone.

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.

Conjugative transfer of high-level mupirocin resistance and the mobilization of non-conjugative plasmids in Staphylococcus aureus.

A 31-kb conjugative plasmid, pXU12, encoding high-level mupirocin resistance via the mupA gene, was isolated from a multiply resistant Staphylococcus aureus isolate, MB494. pXU12 was derived by a deletion of an 8.6-kb EcoRI fragment from a approximately 40-kb plasmid in the parental isolate during curing and conjugation experiments. It transferred rapidly in conjugation experiments, with transconjugants being obtained after 15 min of mating, and mobilized a 3.0-kb erythromycin resistance plasmid, pXU13, from the parental isolate at high frequencies. The cotransfer of pXU13 by pXU12 was unaffected by varying the donor-recipient ratios in the mating mixtures or the length of incubation. pXU12 also mobilized 11 other nonconjugative plasmids belonging to different incompatibility groups and cotransferred at high frequencies. The ability of pXU12 to mobilize different nonconjugative plasmids suggested that it can be used to transfer and isolate non-conjugative plasmids from resistant S. aureus strains in the laboratory, especially from strains where phage-dependent methods of transfer are not applicable.

Molecular fingerprinting of mupirocin-resistant methicillin-resistant Staphylococcus aureus from a burn unit.

OBJECTIVES: To characterize mupirocin-resistant methicillin-resistant Staphylococcus aureus (MRSA) isolated from patients in a burn unit by pulsed-field gel electrophoresis and plasmid contents. METHODS: A total of 53 methicillin-resistant S. aureus, consisting of 48 mupirocin-resistant and 5 mupirocin-susceptible MRSA were compared by plasmid content and pulsed-field gel electrophoresis of Sma I digested genomic DNA. RESULTS: Of the 48 mupirocin-resistant isolates, 39 expressed high-level, and 9 expressed low-level mupirocin resistance. Plasmids were detected in all of the 53 isolates; however, only the high-level mupirocin-resistant isolates contained a 38 kb-conjugative plasmid that encoded high-level mupirocin resistance. Pulsed-field gel electrophoresis divided the isolates into four patterns designated types I to IV. Forty-three isolates consisting of 34 high-level, 5 low-level mupirocin-resistant and 4 mupirocin-susceptible isolates defined the type-I pattern. Eight isolates, five high-level and three low-level mupirocin-resistant isolates had the type-II pulsed-field pattern. The type-III and type-IV pulsed-field patterns consisted of a single isolate each. The type-I and type-II pulsed-field patterns were related and only differed by four Sma I bands. CONCLUSIONS: Results of typing the mupirocin-resistant MRSA from the burn unit with pulsed-field gel electrophoresis indicated that closely related MRSA clones previously circulating in the unit had acquired a high-level mupirocin-resistant plasmid, and spread aided by mupirocin use.

Conjugative transfer of high-level mupirocin resistance from Staphylococcus haemolyticus to other staphylococci.

A conjugative plasmid, pXU10, encoding high-level mupirocin resistance was transferred from a Staphylococcus haemolyticus isolate, CN216, to other coagulase-negative staphylococci and a restriction deficient Staphylococcus aureus strain, XU21, but not to clinical isolates or a restriction-proficient laboratory strain (strain WBG541) of S. aureus. However, from XU21 it was cotransferred with a 3.5-kb chloramphenicol resistance plasmid to WBG541. The results demonstrated the ability of pXU10 to mobilize nonconjugative plasmids.

Molecular characterization of epidemic ciprofloxacin- and methicillin-resistant Staphylococcus aureus strains colonizing patients in an intensive care unit.

Eighteen methicillin-resistant Staphylococcus aureus (MRSA) samples isolated from patients and the environment in an intensive care unit (ICU) during a routine surveillance were tested for antimicrobial resistance and typed by pulsed-field gel electrophoresis. Three pulsed-field patterns were observed. Sixteen were ciprofloxacin resistant and had identical pulsed-field patterns. The results suggested that a ciprofloxacin-resistant MRSA clone had contaminated the environment and spread among patients. This study demonstrates the application of infection control surveillance combined with strain typing in detecting MRSA colonization in the ICU where it was not known to exist.

Antimicrobial resistance of coagulase-negative staphylococci from a Kuwait hospital.

This study investigated the incidence of antimicrobial resistance in clinically significant coagulase-negative staphylococci at the Mubarak Al Kabeer Hospital, Kuwait. A total of 104 isolates of coagulase-negative staphylococci consisting of S. epidermidis (67), S. haemolyticus (16), S. saprophyticus (6), S. simulans (2), S. hominis (4), S. albus (2), S. sciuri (3), S. warneri (2), S. capitis (1), and S. xylosus (1) were isolated from clinical specimens over a 6-7 month period and tested for resistance to 22 antibacterial agents and the ability to produce slime. They were all susceptible to vancomycin and mupirocin but intermediate resistance to teicoplanin was detected in seven isolates: 83 and 47.7% were resistant to penicillin G and methicillin, respectively, 57% were resistant to gentamicin, 49.5% to erythromycin, 50.4% to tetracycline, and 52.3% to trimethoprim. Resistance to heavy metals and the nucleic-acid binding compound was also detected. More than half of S. epidermidis, S. saprophyticus, S. simulans, S. hominis, and all of S. haemolyticus were multiply resistant to three or more groups of antibiotics and there was a significant association between slime production and resistance to multiple antimicrobial agents in S. epidermidis. The results revealed a high level of resistance to commonly used agents.