Tackling contamination of the hospital environment by methicillin-resistant Staphylococcus aureus (MRSA): a comparison between conventional terminal cleaning and hydrogen peroxide vapour decontamination.

The hospital environment can sometimes harbour methicillin-resistant Staphylococcus aureus (MRSA) but is not generally regarded as a major source of MRSA infection. We conducted a prospective study in surgical wards of a London teaching hospital affected by MRSA, and compared the effectiveness of standard cleaning with a new method of hydrogen peroxide vapour decontamination. MRSA contamination, measured by surface swabbing was compared before and after terminal cleaning that complied with UK national standards, or hydrogen peroxide vapour decontamination. All isolation rooms, ward bays and bathrooms tested were contaminated with MRSA and several antibiogram types were identified. MRSA was common in sites that might transfer organisms to the hands of staff and was isolated from areas and bed frames used by non-MRSA patients. Seventy-four percent of 359 swabs taken before cleaning yielded MRSA, 70% by direct plating. After cleaning, all areas remained contaminated, with 66% of 124 swabs yielding MRSA, 74% by direct plating. In contrast, after exposing six rooms to hydrogen peroxide vapour, only one of 85 (1.2%) swabs yielded MRSA, by enrichment culture only. The hospital environment can become extensively contaminated with MRSA that is not eliminated by standard cleaning methods. In contrast, hydrogen peroxide vapour decontamination is a highly effective method of eradicating MRSA from rooms, furniture and equipment. Further work is needed to determine the importance of environmental contamination with MRSA and the effect on hospital infection rates of effective decontamination.

Validation of the NCCLS proposal to use results only from the first isolate of a species per patient in the calculation of susceptibility frequencies.

OBJECTIVE: A recently proposed guideline from the NCCLS recommends that results only from the first isolate of a species per patient be used in calculation of percentage susceptibilities to antimicrobial agents. Because this is apparently based on the comparison of various calculation methods for results for oxacillin against a fairly small number of isolates of Staphylococcus aureus, we have applied these methods to a wider range of antibiotic/organism combinations. METHODS: Antibiotic susceptibility results from our hospital laboratory database were analysed. Rates of antimicrobial susceptibility were calculated using the various criteria proposed by the NCCLS, including exclusion of results from duplicate isolates and surveillance specimens from the calculations. RESULTS AND CONCLUSION: Analysis of results for methicillin against S. aureus, gentamicin against Klebsiella spp., vancomycin against enterococci (all in-patient specimens), and amoxicillin and cefuroxime against Escherichia coli (general practice specimens) confirm that, if duplicates and surveillance specimens are excluded, results obtained with the various patient- and episode-based methods for the calculation of percentage susceptibility are very similar. Because of its simplicity and non-ambiguity, we agree with the suggestion of the NCCLS group that results for the 'first isolate of a given species per patient per analysis period, irrespective of body site, antimicrobial susceptibility profile or other phenotypic characteristics' should be used in the calculation of susceptibility frequencies.

Antibiotic resistance: effect of different criteria for classifying isolates as duplicates on apparent resistance frequencies.

OBJECTIVE: To investigate the effect of screening specimens and different criteria for exclusion of duplicate isolates when surveillance of antimicrobial resistances is performed. MATERIALS AND METHODS: Trends in resistance were analysed for recent isolates of selected organisms from Guy's and St Thomas' Hospitals with the use of various criteria for the exclusion of duplicates, including time since the last isolate and antibiogram pattern, and the effect of excluding screening specimens. RESULTS: There was a significant difference of about 8% in the apparent frequency of methicillin resistance in Staphylococcus aureus in inpatients if the time limit for duplicates was set at 5 rather than 30 days; it was about 10% if a 5 day limit was compared with a 365 day limit. There was also a significant difference, of 6-10%, in apparent resistance frequencies if isolates from screening specimens were excluded. Apparent gentamicin resistance rates in Klebsiella spp. varied between 11% and 28%, and the number of apparent patient isolates of gentamicinresistant organisms varied by up to 35%, depending on the duplicate exclusion criteria chosen. Effects were smaller, though still significant, for vancomycin resistance in Enterococcus spp. There was little effect for amoxicillin or cefuroxime resistance in Escherichia coli isolates from general practitioners, where the proportion of duplicates was small. CONCLUSION: Improved surveillance of antibiotic resistance is needed. However, care needs to be taken in setting the criteria for classifying isolates as duplicates and in comparing results where these criteria may be different or unknown.

A simultaneous outbreak on a neonatal unit of two strains of multiply antibiotic resistant Klebsiella pneumoniae controllable only by ward closure.

Two aminoglycoside-resistant strains of Klebsiella pneumoniae caused an outbreak on the neonatal unit at St Thomas' Hospital. One, which affected 18 patients, was capsular type K18 and resistant to newer cephalosporins by the production of the extended-spectrum beta-lactamase SHV-2; the other, which colonized four patients, was capsular non-typeable and did not produce extended-spectrum beta-lactamase. Both strains were probably brought into the unit by carrier patients; the probable carrier of the non-typeable strain was transferred from another hospital but was negative on a single admission screen; the probable carrier of the K18 strain was not screened on admission because he had been born at St Thomas', but his mother had been transferred from another hospital. Despite intensive efforts to control the outbreak by standard methods of hand washing, screening, patient isolation and environmental cleaning, a total of 22 neonates on the unit eventually became colonized or infected. One of three patients with bacteraemia died. A small proportion of samples of expressed breast milk, electronic thermometers and oxygen saturation probes were contaminated by the K18 strain and may have contributed to some of the cross-infection, but this did not explain the extent of the outbreak. The outbreak was controlled only by opening a temporary ward for colonized neonates and another for newly born babies, which allowed the closure and cleaning of the main neonatal unit. Multiply antibiotic resistant klebsiellas may be highly epidemic and cause serious, difficult-to-control outbreaks on neonatal units. All patients, regardless of their admission history, should be screened on admission for carriage of multiply resistant enterobacteria by a sensitive method, and units should have plans for temporary ward closure should outbreaks occur.

Interpretative reading: recognizing the unusual and inferring resistance mechanisms from resistance phenotypes.

If isolates are speciated and if a sufficient range of antibiotics is tested, underlying resistance mechanisms can often be inferred from the antibiogram data. This allows: (i) anomalous combinations of phenotype and organism to be reconsidered; (ii) prediction of further antibiotics that deserve testing; and (iii) the suppression of susceptibilities that are anomalous in the light of the inferred mechanism. This 'interpretative reading' is widely undertaken in France but is largely precluded in the UK by limited speciation and the testing of narrow ranges of antibiotics. Nevertheless, UK laboratories should be aware of: (i) grossly anomalous combinations of species and phenotype, demanding reference laboratory confirmation; (ii) useful indicator drugs, where resistance implies a mechanism conferring other resistances that may be less obvious in direct tests; and (iii) antibiotics that are prone to select resistant mutants of particular species during therapy. Details of these combinations of organism and resistance are presented. Relationships between antibiogram and mechanism are also presented to allow full interpretative reading for those testing wide panels of drugs versus speciated isolates.

Construction and characterization of mutants of the TEM-1 beta-lactamase containing amino acid substitutions associated with both extended-spectrum resistance and resistance to beta-lactamase inhibitors.

Extended-spectrum TEM beta-lactamases (ESBLs) do not usually confer resistance to beta-lactamase inhibitors such as clavulanate or tazobactam. To investigate the compatibility of the two phenotypes we used site-directed mutagenesis of the bla(TEM-1) gene to introduce into the TEM-1 beta-lactamase amino acid substitutions that confer the ESBL phenotype: TEM-12 (Arg164-->Ser), TEM-26 (Arg164-->Ser plus Glu104-->Lys), TEM-19 (Gly238-->Ser), and TEM-15 (Gly238-->Ser plus Glu104-->Lys). These were combined with three sets of substitutions that confer inhibitor resistance: TEM-31 (Arg244-->Cys), TEM-33 (Met69-->Leu), and TEM-35 (Met69-->Leu and Asn276-->Asp). Introduction of the Arg244-->Cys substitution gave rise to inhibitor-resistant hybrid enzymes that either lost ESBL activity (TEM-12, TEM-15, and TEM-19) or had reduced activity (TEM-26) against ceftazidime. In contrast, the introduction of Met69-->Leu or Met69-->Leu plus Asn276-->Asp substitutions did not significantly affect the abilities of the enzymes to confer resistance to ceftazidime, although increased susceptibility to cefotaxime was observed with Escherichia coli strains that expressed the TEM-19 and TEM-26 beta-lactamases. With the exception of the TEM-12 beta-lactamase, introduction of the Met69-->Leu substitution did not give rise to enzymes with increased resistance to clavulanate compared to that of the TEM-1 beta-lactamase. However, introduction of the double substitution Met69-->Leu plus Asn276-->Asp in the ESBLs did give rise to low-level (TEM-19, TEM-15, and TEM-26) or moderate-level (TEM-12) clavulanate resistance. None of the hybrid enzymes were as resistant to clavulanate as the corresponding inhibitor-resistant TEM beta-lactamase mutant, suggesting that active-site configuration in the ESBLs limits the degree of clavulanate resistance conferred.

Carbapenem resistance in Escherichia coli associated with plasmid-determined CMY-4 beta-lactamase production and loss of an outer membrane protein.

Three cefoxitin-resistant Escherichia coli isolates from stool specimens of a patient with leukemia were either resistant, intermediate, or sensitive to imipenem. Conjugation experiments showed that cefoxitin resistance, but not imipenem resistance, was transferable. All isolates were shown by isoelectric focusing to produce two beta-lactamases with isoelectric points of 5.4 (TEM-1, confirmed by sequencing of a PCR product) and >8.5 (consistent with a class C beta-lactamase). The gene coding for the unknown beta-lactamase was cloned and sequenced and revealed an enzyme which had 99.9% sequence identity with the plasmid-determined class C beta-lactamase CMY-2. The cloned beta-lactamase gene differed from blaCMY-2 at one nucleotide position that resulted in an amino acid change, tryptophan to arginine at position 221. We propose that this enzyme be designated CMY-4. Both the imipenem-resistant and -intermediate isolates lacked a 38-kDa outer membrane protein (OMP) that was present in the imipenem-sensitive isolate. The lack of an OMP alone did not explain the difference in carbapenem susceptibilities observed. However, measurement of beta-lactamase activities (including measurements under conditions where TEM-1 beta-lactamase was inhibited) indicated that the imipenem-intermediate isolate expressed six- to eightfold less beta-lactamase than did the other isolates. This study illustrates that carbapenem resistance in E. coli can arise from high-level expression of plasmid-mediated class C beta-lactamase combined with an OMP deficiency. Furthermore, in the presence of an OMP deficiency, the level of expression of a plasmid-mediated class C beta-lactamase is an important factor in determining whether E. coli isolates are fully resistant to carbapenems.

An outbreak of extended-spectrum, beta-lactamase-producing Salmonella senftenberg in a burns ward.

A strain of Salmonella senftenberg resistant to ceftazidime, gentamicin, chloramphenicol and ciprofloxacin was isolated from burn wounds of eight patients on a burns ward of a hospital in Delhi, India. The organism, which had probably been spread from patient to patient on staff hands, produced the extended-spectrum beta-lactamase SHV-5 and the aminoglycoside-modifying enzymes AAC(3)II + AAC(6'). The strain was not isolated from stool cultures of any of the patients or staff, apart from the index patient who had a history of diarrhoea and fever before admission. The outbreak ended in three weeks, after the implementation of strict handwashing. This is the first report of SHV-5 beta-lactamase in Salmonella spp. and also the first report of SHV-5 in India. The extended-spectrum beta-lactamases that have been reported in Salmonella spp. now include the Group 2 be enzymes SHV-2, SHV-5, TEM-3, TEM-25, TEM-27, CTX-M2, PER-1 and PER-2, and the Group 1 enzymes DHA-1 and CMY-2. The types of extended-spectrum beta-lactamases produced by salmonellas, their association with aminoglycoside resistance and their geographical distribution are now similar to those seen in klebsiella. Increasing antibiotic resistance in these organisms is reducing therapeutic options for the treatment of invasive disease.

Hospital outbreak of Klebsiella pneumoniae resistant to broad-spectrum cephalosporins and beta-lactam-beta-lactamase inhibitor combinations by hyperproduction of SHV-5 beta-lactamase.

An aminoglycoside- and ceftazidime-resistant strain of Klebsiella pneumoniae K2 producing the extended-spectrum beta-lactamase SHV-5 infected or colonized 14 pediatric patients at Guy's Hospital. The patients were mostly neonates recovering from cardiac surgery for congenital defects. The organism was also isolated from a nurse and from the father of one of the children. Four patients had septicemia, and two septicemic neonates with postoperative renal failure died. Aminoglycoside and cephalosporin resistance transferred to Escherichia coli in vitro on a 160-kb plasmid, and a similar resistant E. coli strain was isolated from the stools of one of the affected children. The epidemic organism colonized the bowel and skin and was probably transmitted via staff hands. Five wards were involved because of extensive patient movements. The outbreak was controlled by patient isolation and attention to handwashing. All of the isolates of the outbreak strain were identical by phage typing, ribotyping, plasmid profiling, and biochemical and serological testing, but they varied in their production of SHV-5. Some isolates produced normal amounts of SHV-5 and were susceptible to beta-lactam-beta-lactamase inhibitor combinations. Others, including the single isolate of multiresistant E. coli, produced up to five times as much enzyme as "normal" isolates. This hyperproduction resulted in increased resistance to several penicillins and cephalosporins and to the beta-lactam-beta-lactamase inhibitor combinations amoxicillin-clavulanic acid, ampicillin-sulbactam, piperacillin-tazobactam, and ceftazidime-clavulanic acid. The hyperproduction of SHV-5 by K. pneumoniae and E. coli seen in this outbreak suggests that beta-lactam-beta-lactamase inhibitor combinations may be unreliable for the treatment of organisms producing extended-spectrum beta-lactamases.

Importance of organisms producing broad-spectrum SHV-group beta-lactamases into the United Kingdom.

We report the isolation of three strains of Klebsiella pneumoniae sensu lato, brought into the United Kingdom by patients from Greece and Egypt, that showed plasmid-determined resistance to ceftazidime. All three produced beta-lactamases shown by DNA hybridisation studies to belong to the SHV group. One produced an enzyme that appeared to be related to, but distinguishable from, SHV-1; another SHV-2 and the third SHV-5.

Apramycin and gentamicin resistance in Escherichia coli and salmonellas isolated from farm animals.

Since the aminoglycoside antibiotic apramycin was licensed for veterinary use in 1980, all isolates of Escherichia coli and salmonellas received at the Central Veterinary Laboratory have been monitored for resistance to apramycin and the related antibiotic gentamicin. During the period 1982-4, the incidence of resistance in E. coli to apramycin increased from 0.6% in 1982 to 2.6% in 1984. In salmonellas the incidence of resistance to apramycin increased from 0.1% in 1982 to 1.4% in 1984. Resistance to both apramycin and gentamicin was detected in six different salmonella serotypes, although an isolate of Salmonella thompson from poultry was resistant to gentamicin but not apramycin. Most of the cultures were isolated from pigs, although the incidence of apramycin resistance in S. typhimurium (DT 204C) from calves has shown a recent dramatic increase. All the isolates with one exception produced the enzyme aminoglycoside 3-N-acetyltransferase IV (ACC(3)IV). The resistance was transferable by conjugation in most of the strains examined, and the plasmids specifying the resistance have been found to belong to a number of different incompatibility groups. Plasmids from three E. coli strains were compatible with all the reference plasmids and belonged to a previously undescribed group which was investigated further. It is suggested that bacteria from humans should be examined for resistance to apramycin and gentamicin to determine the possibility of the antibiotic-resistance bacteria, and their genes, spreading from animals to humans.

Resistance to apramycin in Escherichia coli isolated from animals: detection of a novel aminoglycoside-modifying enzyme.

The mechanisms of resistance to apramycin of five isolates of Escherichia coli from animals were investigated. Three isolates, which were resistant to all the aminoglycosides tested, did not transfer their resistance and did not produce aminoglycoside-modifying enzymes. The fourth isolate, which was resistant to apramycin, tobramycin, gentamicin, kanamycin and neomycin but not to amikacin, owed its resistance to production of the acetyltransferase AAC(3)IV. The gene specifying this enzyme was carried on a transposon, Tn800, on a plasmid designated R1535. The fifth isolate was resistant to apramycin, neomycin and kanamycin but not to gentamicin, tobramycin or amikacin. It produced an acetyltransferase that readily acetylated only apramycin, neomycin and paromomycin, a compound that is closely related to neomycin. Synthesis of this enzyme was specified by a chromosomal gene located near pyrD at about 20 min on the map of the E. coli K12 chromosome.

Spectinomycin resistant gonococci.

A study was conducted examining the properties of 10 clinical isolates of spectinomycin resistant gonococci from patients attending clinics at St Mary's and St Thomas's Hospitals, London. All of the isolates produced beta-lactamase and contained plasmids of 2.6, 4.4, and 24.5 megadaltons and required proline for growth. None produced aminoglycoside modifying enzymes. Resistance to spectinomycin was transferred from some of the isolates by transformation but at a much lower frequency than resistance to streptomycin. The isolates from St Mary's Hospital were detected after therapy with spectinomycin, whereas those from St Thomas's Hospital were not. Four recent non-beta-lactamase producing gonococci isolated at St Mary's Hospital and two isolated at St Thomas's Hospital also were not related to use of spectinomycin.

In-vitro antigonococcal activity of rosoxacin (WIN 35213).

The in-vitro activity of rosoxacin against 173 isolates of Neisseria gonorrhoeae, including 17 beta-lactamase-producers, was tested by an agar dilution method. Of the isolates, 167 (including 16 of the beta-lactamase-producers) were inhibited by 0 . 06 mg/l of rosoxacin. The remaining six isolates, one of which produced beta-lactamase and the others were moderately resistant to penicillin, were inhibited by 0 . 12-0 25 mg/l of the compound. There was little correlation between the minimum inhibitory concentrations (MICs) of rosoxacin and penicillin, except for isolates with MICs of penicillin of 0 . 06-1 mg/l, for which correlation was good.

Distribution of genes for trimethoprim and gentamicin resistance in bacteria and their plasmids in a general hospital.

The incidence of trimethoprim resistance in enterobacteria causing infection in a London hospital increased from 5.6% in 1970 to 16% in 1979. The proportion of gentamicin-resistant aerobic Gram-negative bacilli had risen to 6.5% by 1979. During a 5-month period in 1977, during which no epidemic was recognized, all isolates resistant to either trimethoprim, gentamicin, tobramycin or amikacin were studied. The proportion of enterobacteria resistant to both trimethoprim and gentamicin (3.8% of the total) was significantly higher than expected assuming no correlation between acquisition of resistance characters. The resistance was transferable in 23% of trimethoprim-resistant and 76% of gentamicin-resistant strains. Trimethoprim resistance was carried by plasmids of seven different incompatibility groups and in at least four instances was part of a transposon. Gentamicin resistance was determined by plasmids of three groups - IncC, IncFII and IncW. Transposition of gentamicin resistance was not shown, though this may have been the means of evolution of the gentamicin R plasmids of InW, which determined aminoglycoside acetyltransferase, AAC(3). Some bacterial strains with their plasmids were endemic. There was evidence for these plasmids (i) acquiring new resistance genes by transposition, (ii) losing resistance genes by deletion and (iii) being transferred between bacterial species in the hospital.