Dissemination of methicillin-resistant Staphylococcus aureus SCCmec type IV and SCCmec type V epidemic clones in a tertiary hospital: challenge to infection control.

Two-hundred MRSA strains from inpatients with healthcare-associated (HA) and 100 MRSA strains from outpatients with community-associated (CA) skin and soft tissue infections (SSTIs) were tested for antimicrobial susceptibility, staphylococcal cassette chromosome mec (SCCmec) typing, Panton-Valentine leucocidin (PVL) toxin, seh and arcA genes. Based on SCCmec typing, HA-MRSA isolates were further divided into HA-SCCmec I/II/III MRSA and HA-SCCmec IV/V MRSA, and CA-MRSA isolates into CA-SCCmec I/II/III MRSA and CA-SCCmec IV/V MRSA. SCCmec types were further characterized by pulsed-field gel electrophoresis, spa typing and multi-locus sequence typing. Seventy-five (37·5%) HA-MRSA isolates and 83/100 CA-MRSA isolates were SCCmec IV/V genotype. HA-SCCmec IV/V MRSA was associated with malignancy (P = 0·03) and bone fractures (P = 0·02) compared to CA-SCCmec IV/V MRSA. HA-SCCmec IV/V MRSA was associated with PVL gene carriage compared to HA-SCCmec I/II/III MRSA (P < 0·001). ST22-MRSA-IV (EMRSA-15), ST772-MRSA-V, and ST36-MRSA-IV and ST239:EMRSA-I:III were the major clones identified. Our study documents the emergence of SCCmec IV and SCCmec V MRSA clones in an Indian hospital.

A case of community-onset meningitis caused by hospital methicillin-resistant Staphylococcus aureus successfully treated with linezolid and rifampicin.

OBJECTIVE: To report a relatively rare presentation of methicillin-resistant Staphylococcus aureus (MRSA) meningitis in a previously healthy boy in Kuwait. CLINICAL PRESENTATION AND INTERVENTION: A 14-year-old boy presented with a 2 weeks' history of headache and fever with increasing severity. He developed photophobia and double vision 2 days prior to his hospital visit and received ceftriaxone for 6 days prior to admission to the hospital. There was no history of head trauma or neurosurgical operation. Lumbar puncture revealed a slightly turbid cerebrospinal fluid with pleocytosis and greatly reduced glucose, elevated protein level and on culture grew MRSA. Staphylococcal chromosome cassette mec (SCCmec) typing revealed that it belonged to SCCmec type III and sequence type 238 (ST238-SCCmec-III). Polymerase chain reaction screening for the presence of Panton-Valentine leukocidin (PVL) genes yielded a negative result; all these findings were consistent with hospital-acquired MRSA. He was treated with intravenous linezolid and rifampicin for 2 weeks, made good response and was discharged home fully recovered and well. CONCLUSION: Hospital MRSA should be considered in the differential diagnosis of the causative agents of community-onset meningitis in healthy patients even without predisposing factor.

Genetic lineages of community-associated methicillin-resistant Staphylococcus aureus in Kuwait hospitals.

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.

Surveillance of antibacterial resistance in Staphylococcus aureus isolated in Kuwaiti hospitals.

OBJECTIVE: To investigate the prevalence of antibiotic resistance among Staphylococcus aureus isolated in Kuwaiti hospitals. MATERIALS AND METHODS: S. aureus were isolated and identified following standard microbiological methods. Antibacterial susceptibility test was performed by disk diffusion and the measurement of minimum inhibitory concentration with E-test strips. RESULTS: A total of 1,846 S. aureus isolates were analyzed from 13 hospitals between 1 March and 30 October 2005. They were isolated from 1,765 (95.6%) inpatients and 81 (4.4%) outpatients. Methicillin resistance was detected in 588 (32.0%) of the isolates. The methicillin-resistant S. aureus (MRSA) consisted of 461 (78%) multiresistant and 127 (22%) nonmultiresistant isolates. The nonmultiresistant MRSA consisted of epidemic MRSA-15 and community-associated MRSA. The community-associated MRSA was detected in all hospitals with MRSA, indicating its establishment in Kuwaiti hospitals. The proportion of isolates resistant to gentamicin, kanamycin, erythromycin, tetracycline, ciprofloxacin, fusidic acid and trimethoprim was higher among MRSA than methicillin-susceptible S. aureus (MSSA) isolates. Twenty-four and 22% of MRSA and MSSA isolates, respectively, expressed reduced susceptibility to vancomycin (minimum inhibitory concentration = 3-4 mg/l). CONCLUSION: The study revealed the presence of methicillin resistance in 32% of S. aureus isolated in Kuwaiti hospitals and revealed an increase in the number of MRSA and MSSA with reduced susceptibility to vancomycin.

Characterisation of non-multiresistant methicillin-resistant Staphylococcus aureus (including EMRSA-15) in Kuwait Hospitals.

This study characterised non-multiresistant methicillin-resistant Staphylococcus aureus (nmMRSA) isolates from Kuwait hospitals to ascertain whether they were community-acquired MRSA (CA-MRSA). Forty-two nmMRSA isolates obtained between July 2001 and October 2003 were analysed by staphylococcal cassette chromosome mec (SCCmec) typing, bacteriophage typing, production of Panton-Valentine leukocidin (PVL), urease and staphylococcal enterotoxins A, B, C and D, TSST-1, and by pulsed-field gel electrophoresis (PFGE). Forty-one isolates were SCCmec type IV, and one isolate was SCCmec type III. The isolates belonged to six PFGE patterns, with two types, A and D, distributed in six and four hospitals, respectively. Most (n = 26; 61.9%) isolates produced urease. These isolates were mainly from wound and skin infections, showed low-level methicillin resistance (MIC 8-48 mg/L), and nine carried genes for PVL. These characteristics, together with their carriage of the type-IV SCCmec, identified the isolates as CA-MRSA. Ten of the 16 urease-negative isolates produced staphylococal enterotoxin C; 12 reacted weakly with phage 75, and were resistant to clindamycin and/or erythromycin, which are characteristics of EMRSA-15. Thus, this study identified the co-existence of two types of nmMRSA, i.e., CA-MRSA and EMRSA-15, in Kuwait hospitals.

High prevalence of toxic shock syndrome toxin-producing epidemic methicillin-resistant Staphylococcus aureus 15 (EMRSA-15) strains in Kuwait hospitals.

This study characterized EMRSA-15 isolates obtained from patients in Kuwait hospitals for their genotypic relatedness, antibiotic resistance and carriage of virulence genes using pulsed-field gel electrophoresis (PFGE), coagulase serotyping, SCCmec subtyping, spa typing, multilocus sequence typing and DNA microarray. The isolates were resistant to trimethoprim (75.6%), ciprofloxacin (29.7%), erythromycin and clindamycin (24.3%), tetracycline (19.0%), and gentamicin and kanamycin (21.6%). All 37 isolates belonged to sequence type (ST) 22, coagulase type XI, three PFGE types and eight subtypes, ten spa types including t223 (51.3%), t852 (13.5%), t032 (8.1%), t790 (8.1%), t3107 (5.4%) and one each of t309, t2251, t3935, t5708 and t5983. Twenty-six isolates (70.2%) carried SCCmec IVa, eight isolates carried SCCmec IV and three isolates carried SCCmec IVh. All isolates carried agr1, cap5 and egc gene cluster (seg, sei, selm, seln, selo, and selu). tst (toxic shock syndrome toxin) was detected in 23 isolates. Eight isolates (21.6%) were positive for Panton-Valentine leukocidin (PVL). Genotypic analysis revealed that 62.1% of the isolates comprising ST22-IVa-t223 (51.3%) and ST22-IVa-t309/t2251/t3935/t5708 (10.8%) were CC22-[tst1(+)] UK EMRSA-15/Middle Eastern variant, 21.6% were CC22-PVL(+) EMRSA-15 variant and 16.2% were CC22-UK EMRSA-15/Barnim clone. These results show that the tst1 positive-ST22-IVa-t223 (Middle Eastern variant) and the CC22-PVL(+) EMRSA-15 variant were the dominant EMRSA-15 variants in Kuwait hospitals.

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.

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.

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.

Antibiotic resistance of enterococci isolated at a teaching hospital in Kuwait.

Enterococci isolated in a teaching hospital were studied for their resistance to different antibiotics. Minimum inhibitory concentrations to high-level aminoglycosides and glycopeptide antibiotics were determined by agar dilution and E-test methods respectively. Genes encoding aminoglycoside-modifying enzymes were detected by the polymerase chain reaction (PCR). 195 enterococci were isolated from urines (54.3%), wounds (16.4%), blood (10.2%), and miscellaneous sources (18.9%). They consisted of E. faecalis (88.7%), E. faecium (9.2%), E. casseliflavus (1.5%) and E. bovis (0.5%). None of the enterococci produced penicillinase but 3.5% of them were resistant to ampicillin. They were also resistant to high-level gentamicin (15.9%), kanamycin (22.0%), streptomycin (21.0%), tetracycline (65.1%), erythromycin (62.6%), ciprofloxacin (36.1%), chloramphenicol (26.1%), vancomycin (3.0%) and teicoplanin (2.0%). Most of the high-level aminoglycoside-resistant isolates contained genes coding the bifunctional aminoglycoside modifying enzymes AAC(6')-APH(2"), APH(3') and ANT(6') but not the ANT(4') enzyme. The results demonstrated a low prevalence of vancomycin resistance among Enterococci in this hospital.

A new incompatibility group plasmid in Staphylococcus aureus.

The conjugative plasmid pWBG637 and its derivatives, pWBG636 and pWBG642, were tested for incompatibility against conjugative and non-conjugative plasmids in Staphylococcus aureus. They were compatible with other conjugative plasmids and plasmids of the 14 established incompatibility groups. They therefore define a new Incompatibility group 15.

Conjugal transfer of plasmid pWBG637 from Staphylococcus aureus to Staphylococcus epidermidis and Streptococcus faecalis.

Plasmids pWBG636 (GmR) and pWBG642 (EmR) derived from the conjugative plasmid pWBG637 were tested for ability to transfer conjugatively to Staphylococcus epidermidis, Streptococcus faecalis, Bacillus subtilis and Escherichia coli. Both plasmids transferred to S. epidermidis and Strept. faecalis but not to B. subtilis and E. coli. Once in S. epidermis and Strept. faecalis they were transferred back to S. aureus. Restriction endonuclease analysis showed that the plasmids were stably maintained in S epidermidis and Strept. faecalis.

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.

Typing of methicillin-resistant Staphylococcus aureus with IS256.

A simple one-step procedure is described for specifically amplifying and labelling insertion element IS256 which is associated with the gentamicin-resistance transposon Tn4001. The product has been used to probe DNA digests of methicillin-resistant Staphylococcus aureus. The resulting restriction fragment length polymorphisms were found to be able to distinguish isolates which were indistinguishable by other typing methods. The probe also hybridised with methicillin-resistant Staphylococcus aureus which were isolated before the emergence of gentamicin resistance, demonstrating its usefulness in typing species other than those that are gentamicin-resistant.

Conjugative trimethoprim resistance in Staphylococcus aureus.

A multiply resistant Staphylococcus aureus isolate, WBG7410, harbours plasmids of 38, 26, 2.8, 2.4 and 1.9 kb and transfers trimethoprim and kanamycin resistance at high frequencies by conjugation. The transconjugants contained the 38-kb plasmid, pWBG707, and the 2.8-kb plasmid. Plasmid pWBG707 was shown to encode trimethoprim resistance, was conjugative and mobilised at high frequencies the 2.8-kb plasmid which presumably encodes kanamycin resistance. Plasmid pWBG707 was isolated mostly in the open circular form and analysis with EcoRI restriction endonuclease suggests that pWBG707 is a new conjugative plasmid distinct from the other conjugative plasmids reported in S. aureus.

A new class of conjugative plasmid in Staphylococcus aureus.

Plasmid pWBG637, a Staphylococcus aureus conjugative plasmid having no known resistance phenotype, was compared with other conjugative plasmids in S. aureus by restriction endonuclease analysis, incompatibility testing and DNA-DNA hybridisation. It differed from the other conjugative plasmids on all three criteria and thus belongs to a new class of conjugative plasmids.

Transfer of resistance determinants from a multi-resistant Staphylococcus aureus isolate.

The clinical isolate Staphylococcus aureus WBG1024 was resistant to cadmium, benzyl penicillin, kanamycin, neomycin, streptomycin, tetracycline and trimethoprim and harboured a conjugative plasmid pWBG637 (34.5 kb) and non-conjugative plasmids of 23.8, 4.4, 2.8 and 1.9 kb. Transduction and mixed-culture transfer experiments demonstrated that the 4.4-kb plasmid (pWBG632) encoded resistance to tetracycline and the 23.8-kb plasmid (pWBG628) encoded resistance to cadmium, benzyl penicillin, kanamycin, neomycin and streptomycin. The conjugative plasmid pWBG637 was able to mobilise a further 4.4-kb plasmid (pWBG633) encoding streptomycin resistance and recombined with the multiresistance plasmid pWBG628 to produce transconjugantes of various resistance phenotypes.

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.

Excision of a conjugative plasmid from the staphylococcal chromosome.

Staphylococcus aureus isolate WBG1003 resistant to benzyl penicillin, cadmium, arsenate and streptomycin harbours two plasmids of 38.8 (pWBG621) and 4.4 (pWBG625) kb. In conjugation experiments two types of streptomycin-resistant transconjugants were obtained; one carried a 4.4-kb plasmid and the other, a 34.5-kb and a 4.4-kb plasmid. The 34.5-kb plasmid (pWBG620) has been found to be conjugative and able to mobilise non-conjugative plasmids. It has no detectable resistance phenotype and has not been detected in WBG1003 nor in the recipient used in the conjugation experiments. Restriction endonuclease analysis and DNA-DNA hybridisation have revealed that pWBG620 is unrelated to pWBG621 present in strain WBG1003. The data presented indicate that pWBG620 is in the chromosome of strain WBG1003 and that it excises during conjugation.

Genetic analysis of methicillin-resistant Staphylococcus aureus from a Nigerian hospital.

Methicillin-resistant strains of Staphylococcus aureus isolated during 1985 and 1987 at a Nigerian hospital were compared by resistance profiles, plasmid analysis, and pulsed-field gel electrophoresis of chromosomal DNA. The results indicated that the isolates from the two periods were unrelated with regard to all three aspects. None of the isolates was similar to the classical MRSA nor to the epidemic MRSA of Australia or the UK. The MRSA isolated in 1985 had a similar plasmid to MRSA isolates from Singapore, but differed from them when compared by pulsed-field gel electrophoresis.