a Department of Medical Microbiology; and b Pathology Computing Department, City Hospital NHS Trust, Birmingham B18 7QH, UK
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ten Teaching (T), ten Associate Teaching (AT) and ten District General (DG) Hospitals in the UK were enrolled into the study. Selection was determined by type of hospital and geographical location. Centres were asked to save consecutive, clinically significant isolates, comprising 60 S. aureus, 60 coagulase-negative staphylococci (CNS), 40 pneumococci and 20 enterococci, from the beginning of October 1996 to the end of January 1997. Centres were asked to exclude duplicate isolates from the same patient and multiple isolates of S. aureus originating from a cross-infection source. Isolates and details of site of isolation were sent by the participating centres to a central laboratory for further study.
Identification
Identification of the isolates was confirmed by the central laboratory using optochin susceptibility for pneumococci; Pastorex and DNase testing for S. aureus; Pastorex, DNase and ID32 Staph (bioMérieux, Basingstoke, UK) for CNS; and Rapid ID32 Strep (bioMérieux) for enterococci. Once identification was confirmed, isolates were suspended in 13% glycerol broth and stored on beads at -70°C.
Minimum inhibitory concentrations (MICs)
MICs were determined with methodology based on that recommended by the British Society for Antimicrobial Chemotherapy (BSAC). 7 Briefly, Iso-Sensitest agar (ISTA; Oxoid, Basingstoke, UK) was used as basal medium (supplemented with 5% whole horse blood for testing pneumococci only), except when determining the sensitivity of staphylococci to methicillin and oxacillin. In this instance, Mueller-Hinton agar (Difco Laboratories, West Molesey, UK) was used. Overnight broth cultures or organism suspensions were diluted in sterile distilled water to yield, after inoculation with a multipoint inoculator (Mast Diagnostics, Bootle, UK), a final inoculum of 10 4 cfu/spot. For testing methicillin and oxacillin a heavier inoculum of 10 6 cfu/spot was employed. Plates were incubated at 35-37°C for 18-20 h in air. The atmosphere was enriched with 4-6% CO 2 for pneumococci and a temperature of 30°C was used for the detection of resistance to methicillin and oxacillin in staphylococci. The MIC was defined as the lowest concentration of antimicrobial inhibiting growth, one or two colonies being ignored. Control isolates S. pneumoniae ATCC 49619, S. aureus ATCC 25923, S. aureus NCTC 6571 and Enterococcus faecalis ATCC 29212 were included with every batch of MIC determinations.
Standard antimicrobial powders for determining MICs were obtained from the following sources: dalfopristin- quinupristin, Rhône- Poulenc Rorer (Paris, France); penicillin, methicillin and mupirocin, SmithKline Beecham (Worthing, UK); ciprofloxacin, Bayer AG (Wuppertal, Germany); clindamycin, Upjohn (Crawley, UK); erythromycin, Abbott Laboratories (Maidenhead, UK); fusidic acid, Leo Laboratories (Aylesbury, UK); gentamicin, cefotaxime and teicoplanin, Hoechst Marion Roussel (Uxbridge, UK); chloramphenicol, rifampicin, doxycycline and nitrofurantoin, Sigma Chemicals (Dorset, UK); vancomycin, Lilly Industries (Basingstoke, UK); tetracycline, Cyanamid (Pearl River, NY, USA). All antimicrobials were prepared on the day of MIC testing, except for dalfopristin- quinupristin, where aliquots of a stock 100,000 mg/L solution were stored at -70°C (frozen and thawed once and then discarded).
Detection of reduced sensitivity to vancomycin in S. aureus
It has been reported by the Public Health Laboratory Service that isolates with homogeneous resistance to vancomycin, i.e. MICs of vancomycin of 8 mg/L, would be detected using a conventional method of testing similar to that previously described. 8 However, for those isolates with heterogeneous resistance, that is, isolates with vancomycin MICs 2-8 mg/L which contain sub-populations with MICs of vancomycin 8 mg/L, a one-step selection with vancomycin should be used. A screening method devised by K. Hiramatsu (personal communication) was therefore used. Briefly, isolates were grown overnight in Tryptone Soya Broth (Oxoid) and adjusted to an optical density of 0.15 at a wavelength of 540 nm in sterile distilled water, then 10 µL of this suspension was inoculated on to the surface of a Brain Heart Infusion Agar (BHIA, Oxoid) plate containing vancomycin 4 mg/L. After 48 h incubation at 35-37°C, any colonies growing on the surface were subcultured into broth for conventional agar dilution MIC determination on ISTA, on ISTA containing 2% lysed horse blood and on BHIA, with inocula of 10 4 and 10 6 cfu/spot. MICs were also determined by Etest (CDS, Cambridge, UK) following the manufacturer's instructions. All plates were incubated at 35-37°C in air. S. aureus isolates Mu3 (heterogeneous resistant isolate) and Mu50 (homogeneous resistant isolate) were isolated in Japan and were obtained from Professor K. Hiramatsu. S. aureus isolates ATCC 25923 and NCTC 6571 were included as controls.
Detection of ß-lactamase in enterococci
Blotting paper strips soaked with nitrocefin reagent (Oxoid) were streaked with pure cultures of the enterococci resistant to ampicillin. Strips were incubated at 37°C for 10 min after which time, if there was no colour change, tests were considered negative. ß-Lactamase-positive isolates were included as controls.
Interpretation of sensitivity
An organism was designated sensitive if its MIC was greater than or equal to the in-vitro
breakpoint concentration (BP). For the majority of the antimicrobials BPs were taken from the
BSAC Guidelines.
7 However, for some antimicrobials data were not available
in the BSAC document. For doxycycline and oxacillin the recommendations of the
Comité de l'Antibiogramme de la Société Française de
Microbiologie
9 were used, and for mupirocin resistance in staphylococci
and high-level gentamicin resistance in enterococci the recommendations of Gilbart et al.
10 and the National External Quality Assessment Scheme
for Microbiology,
11 respectively, were used. The National Committee for
Clinical Laboratory Standards (NCCLS) suggests BPs for interpretation which are slightly
different from those of the BSAC
12 for penicillin (susceptible 0.06 mg/L, intermediate
0.1- 1 mg/L and resistant
2 mg/L) and for cefotaxime (susceptible
0.5 mg/L,
intermediate 1 mg/L and resistant
2 mg/L). The data for penicillin and cefotaxime were
analysed using both interpretative criteria.
With dalfopristin- quinupristin a tentative BP of 2 mg/L was used, this concentration being suggested by Rhône Poulenc Rorer after review of world-wide MIC distributions and clinical response data (H. Nadler, M. Dowzicky, G. Talbot, F. Bayport & M. Pease, personal communication).
Statistical analysis
The level of agreement between methicillin and oxacillin BPs in detecting methicillin resistance (Student's paired t-test) and differences in rate of resistance for each of the organism- antibiotic combinations between the three types of hospital (Kruskal-Wallis non-parametric one-way analysis of variance) were determined statistically using a standard statistical package (Instat, GraphPad Software Inc., San Diego, CA, USA).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Compared with the numbers of isolates requested, 96.9% S. aureus, 86.3% CNS, 86.7% S. pneumoniae and 94.3% enterococci were received. Only 334 isolates (55.7% requested isolates) of CNS were received from the DG hospitals, this being somewhat less than the numbers received from the other two types of hospital (97.3% and 77.2% requested isolates for T and AT hospitals, respectively). However, this was not unexpected, reflecting the types of infection encountered in these institutions.
For staphylococci, little difference between the three types of hospital was observed in percentage of isolates from the various sites (70-80% of isolates being from wounds), except for CNS isolated from urines, lines and tips. Nine per cent of CNS isolates collected by the T hospitals were from urines compared with 45% and 37%, respectively, for AT and DG hospitals. Thirty-three per cent, 18% and 19% CNS were isolated from lines and tips at the T, AT and DG hospitals, respectively. Of the CNS isolates the majority were identified as Staphylococcus epidermidis (49.5%), Staphylococcus haemolyticus (14%) and Staphylococcus saprophyticus (14%). More than 60% of the enterococci were isolated from urine and 90.6% were identified as E. faecalis. The second most common enterococcal species isolated was Enterococcus faecium (31 isolates), the majority of these isolates being from blood (61%) and urine (29%). With pneumococci, 92%, 84.3% and 83.8% of requested numbers were isolated by the T, AT and DG hospitals, respectively.
MIC determinations with control isolates
MICs for the reference control isolates were within ± one dilution step of expected MICs (data not shown).
Detection of methicillin- oxacillin resistance
Statistically significant differences in resistance rates, depending on whether methicillin MICs and BP (4 mg/L) or oxacillin MICs and BP (2 mg/L) were used, were observed only for S. epidermidis(P <0.0001), S. saprophyticus (P < 0.0001) and other CNS species (P = 0.0112).
Resistance rates
S. aureus.Percentage resistance rates for all of the antibiotics tested are shown in Table I. No resistance to dalfopristin- quinupristin, teicoplanin and vancomycin was seen, and only 0.5% resistance to rifampicin was observed. Amongst these isolates <1% exhibited high-level resistance (MIC >512 mg/L) to the topical antibiotic mupirocin.
|
Variability in resistance rates was more marked for methicillin than for any of the other
antibiotics tested, with rates ranging from 0% to 56.7%. A detailed picture of resistance rate and
location in the UK for each of the types of hospital is shown in Table II. Amongst the T hospitals
resistance rates for methicillin ranged from 1.7% to 35%, with the highest rates observed in
Sheffield (35%) and one of the London hospitals (20%). For the AT hospitals, resistance rates
ranged from 0% to 56.7%, with the highest level of resistance being observed in Londonderry. In
the DG hospitals, levels of resistance ranged from 0% to 56.3%, but five of the hospitals had
rates 3.3%. The highest level of resistance was seen in Wolverhampton (56.3%), and a high
level of resistance was also observed in the AT hospital (Birmingham) in the same region.
|
|
S. haemolyticus.Resistance rates for S. haemolyticus are shown in Table I. All of the isolates were sensitive to dalfopristin- quinupristin and vancomycin. Statistically significant differences were seen between the three types of hospital for ciprofloxacin (P = 0.0169), oxacillin (P = 0.0244) and teicoplanin (P = 0.0244). The significant difference, in all cases, was observed between the T and the AT hospitals (P < 0.05). Resistance rate to teicoplanin, related to geographical location, is shown in Table II.
S. saprophyticus.All of the isolates were sensitive to dalfopristin-quinupristin,
rifampicin and
vancomycin, and resistance rates for clindamycin, teicoplanin and ciprofloxacin were 4%
(Table I). The two isolates resistant to teicoplanin, with MICs of 8 mg/L
and 16 mg/L, were
isolated in London (T) and the West Midlands (DG). No statistically significant difference
between the three types of hospital was observed (P values ranging from 0.1306 to
0.8671), except for oxacillin (P = 0.0357), with which a significant difference
between resistance rates in the AT and DG hospitals was observed (P < 0.05).
CNS (other than S. epidermidis, S. haemolyticus and S. saprophyticus).Three hundred and eleven isolates were identified as staphylococcal species other than epidermidis, haemolyticus and saprophyticus. None of these isolates was resistant to dalfopristin-quinupristin or vancomycin. Of the isolates, 32.6% and 28.0% were resistant to the ß-lactam antibiotics, penicillin and methicillin, respectively, 38.0% to erythromycin, 25.7% to gentamicin, 24.4% to fusidic acid, 10.6% to ciprofloxacin, 5.1% to clindamycin, 2.6% to rifampicin and 1.6% to teicoplanin (one Staphylococcus capitis and four Staphylococcusspp. with MICs of teicoplanin 8 mg/L).
E. faecalis.A summary of resistance rates for this species is shown in Table IV. No statistical difference between the three types of hospital for any of the antibiotics tested was seen (P values ranged from 0.1281 to 0.9953), except for erythromycin (P = 0.0023). In this instance, a significant difference was observed between the rates of resistance in the T and DG hospitals (84.5% and 55.2%, respectively).
|
E. faecium. No significant difference between the resistance rates in the T, AT and DG hospitals was seen for any of the antibiotics tested (P values ranged from 0.1042 to 0.9131). The 31 isolates studied were more sensitive to dalfopristin-quinupristin than wasE. faecalis, with only one isolate having an MIC one dilution above the BP (Table IV). Three isolates resistant to the glycopeptides comprised one resistant to vancomycin (MIC 8 mg/L) and sensitive to teicoplanin, and two (isolated from the same centre) equally resistant to vancomycin and teicoplanin.
Other enterococcal species.The combined resistance rates for the 22 isolates are shown in Table IV. One unspeciated isolate of Enterococcusand one isolate of Enterococcus gallinarum, both isolated at T hospitals, had high-level resistance to gentamicin (MICs 4096 mg/L and 2048 mg/L, respectively) with simultaneously raised MICs of ampicillin (MICs 128 mg/L and 32 mg/L, respectively).
S. pneumoniae.No significant difference in resistance rates between the three types of hospital was observed (P values ranged from 0.1025 to 0.9181) except for erythromycin (P = 0.0424). Only two isolates, from the same DG hospital, were resistant to dalfopristin- quinupristin with MICs of 16 mg/L (Table V). Interestingly, both of these isolates were sensitive to penicillin but were highly resistant to erythromycin (MICs > 128 mg/L).
|
High-level resistance to ciprofloxacin (MIC > 32 mg/L) was detected only in five isolates: two from Cambridge and one each from Cardiff, Manchester and London. However, 6.9% of the total isolates had low-level resistance (MICs 4-8 mg/L). In two centres (Cambridge and Edinburgh) no erythromycin resistance was detected. Of the isolates resistant to penicillin, yet sensitive to cefotaxime, 25.8% were resistant to erythromycin compared with 29.2% for those isolates with intermediate sensitivity to penicillin. The three isolates resistant to both ß-lactam antibiotics were also resistant to erythromycin. No resistance to vancomycin was detected in any of the isolates.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Rates of methicillin resistance amongst the S. aureusinvestigated in this study varied greatly depending on geographical location in the UK, with rates of resistance ranging from 0% to 56.7%. Every effort was made in the design of this study to avoid the inclusion of multiple isolates from the same patient or from a cross-infection episode. However, it is not inconceivable that at the centres with the highest rates of resistance this may have occurred.
In this study resistance rates when testing methicillin and oxacillin were similar for all of the
isolates tested except for S. saprophyticus, S. epidermidis and some of the
other CNS. Interpretation of sensitivity and thus the determination of resistance rates can yield
different rates of resistance depending on the criteria used. When BPs are devised, both
pharmacokinetic properties and in-vitro activity of the antimicrobial agent are considered, with
priority given to those pathogens against which the drug is targeted. However, for antimicrobials
such as methicillin, which is tested as a means of detecting a mechanism of resistance rather than
for clinical use, the BP may be biased towards those organisms which will be most frequently
tested, in this instance S. aureus and S. epidermidis. In this study, with a
methicillin BP of 4 mg/L, 56.7% of S. saprophyticus isolates were designated resistant
to methicillin. The MIC distribution, MIC
50 and MIC
90 values for S. saprophyticus would suggest that isolates with MICs of
methicillin of 8 mg/L, the concentration inhibiting 90% of isolates, are not resistant to this
agent.
In studies characterizing the mechanism and rate of resistance in S. saprophyticus
13
,14
it is suggested that an MIC of methicillin 16
mg/L is a more appropriate breakpoint and correlates with resistance associated with the mecA gene. If this criterion was applied to the data in this study the resistance rate for all of
the isolates would fall from 62.4% to 5.7%. Differences in the other CNS are not so easily
explained, but the information will be valuable when developing methods based on testing
oxacillin.
The emergence of S. aureus isolates with reduced susceptibility to vancomycin has presented technical problems for diagnostic laboratories. Disc sensitivity testing methods fail to detect resistance, 8 but screening methods have been devised to identify the minority population which may have reduced sensitivity. From this study four isolates have been identified which are worthy of further examination by electron microscopy to determine if they have a thickened cell wall typical of vancomycin-intermediate isolates. 15 However, as can be seen from these data, confirmation of resistance by conventional agar incorporation methodology is not always conclusive. Indeed, only when BHIA was employed as growth medium were elevated MICs discernible for the four clinical and Mu3 and Mu50 reference isolates. Further work is therefore needed to explain these findings.
Five hundred and thirteen (90.6%) of the enterococci studied in this survey were identified as E. faecalis. Resistance rates to ampicillin, vancomycin and high-level resistance to gentamicin for these isolates were 1.4% (all ß-lactamase negative), 0.8% and 10.7%, respectively. The rates of resistance to ampicillin and vancomycin are similar to those observed in two recent studies, one looking at enterococci isolated from blood cultures over an 8 year period in Nebraska, 16 the other, a Greek study, looking at consecutive non-repetitive isolates during 1996-7. 17 In these studies, however, rates of high-level resistance to gentamicin were much higher (Nebraska 36.1%, Greece 43.7%). With regard to E. faecium, amongst the three studies there was general agreement in ampicillin resistance rates. However, resistance to vancomycin was more frequent in the Nebraska study (22.2%) compared with 9.7% for this study and 0% in the Greek study. High-level resistance to gentamicin was also greater in the Nebraska and the Greek studies (28.9% and 26.3%, respectively) than the 3.2% in this study.
Rates of resistance in S. pneumoniae varied significantly depending on geographical location. 18 In a UK survey of community-acquired lower respiratory tract pathogens undertaken in the latter part of 1994 and early part of 1995, 19 with amoxycillin MICs used to judge degrees of resistance, 3.5% of the isolates had intermediate sensitivity and 3.3% exhibited HLR. In contrast, if rates of resistance in this study were reviewed by geographical location in the UK, LLR rates varied from 0% (London, Bristol, Glasgow and Belfast) to 10.7% (Nottingham). HLR rates varied from 0% (London, Birmingham, Nottingham, Bangor, Newcastle and Glasgow) to 14% and 11.9%, respectively, in Liverpool and Belfast. Although resistance rates from the 1994-5 study 19 and our survey are not directly comparable, this survey suggests that resistance rates have changed with time. For example, in London, LLR was detected in both T and AT hospitals (3.1% and 2.9%, respectively) and HLR seen in T hospitals (3.1%) in our study, compared with no resistance in the 1994-5 study.
The widespread emergence and distribution of multiple resistant S. aureus (MRSA) has limited the use of the ß-lactam antibiotics. Dalfopristin-quinupristin has been introduced as an alternative therapeutic agent. No resistance to dalfopristin-quinupristin was observed amongst the staphylococci, including those isolates resistant to methicillin, erythromycin and ciprofloxacin. Dalfopristin- quinupristin was also active against the two isolates in Japan with reduced susceptibility to vancomycin and the four clinical isolates with raised MICs of vancomycin when tested on BHIA. Of E. faecalis 35.7% were resistant to dalfopristin- quinupristin, but only one isolate each of E. faecium, E. casseliflavus and an unspeciated enterococcus had MICs of 4 mg/L, a concentration one dilution above the in-vitro BP for dalfopristin-quinupristin. MIC data for all of the isolates tested were in agreement with that published by Schouten & Hoogkamp-Korstanje 20 except for S. pneumoniae, for which isolates with MICs above 0.5 mg/L were not detected in the study of Schouten & Hoogkamp-Korstanje.
This study has confirmed that rates of resistance vary depending on locality in the UK and to a degree, the nature of the hospital. It will be interesting to see if rates of resistance fluctuate during subsequent studies of resistance rates at the same centres.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Voss, A., Milatovic, D., Wallrauch-Schwarz, C., Rosdahl, V. T. & Braveny, I. (1994). Methicillin-resistant Staphylococcus aureus in Europe. European Journal of Clinical Microbiology and Infectious Diseases 13, 505.[ISI][Medline]
3
.
Kunin, C. M. (1993). Resistance to antimicrobial drugsa worldwide
calamity. Annals of Internal Medicine 118, 55761.
4
.
Hiramatsu, K., Hanki, H., Ino, T., Yabuta, K., Oguri, T. & Tenover, F. C. (1997).
Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin
susceptibility. Journal of Antimicrobial Chemotherapy 40, 1356.
5 . Centres for Disease Control. (1997). Staphylococcus aureus with reduced susceptibility to vancomycinUnited States, 1997. MMWRMorbidity and Mortality Weekly Report 46, 7656.
6
.
Bouanchaud, D. H. (1997). In-vitro and in-vivo antibacterial activity of
quinupristin/dalfopristin. Journal of Antimicrobial Chemotherapy 39, Suppl. A, 1521.
7 . Working Party on Antibiotic Sensitivity Testing of the British Society for Antimicrobial Chemotherapy. (1991). A guide to sensitivity testing. Journal of Antimicrobial Chemotherapy 27, Suppl. D, 150.[ISI][Medline]
8 . Anonymous. (1997). Detecting vancomycin intermediate Staphylococcus aureus.Communicable Disease Report CDR Weekly 7, 417, 20.[Medline]
9 . Comité de l'Antibiogramme de la Société Française de Microbiologie. (1996). Clinical Microbiology and Infection2, Suppl. 1.
10 . Gilbart, J., Perry, C. R. & Slocombe, B.(1993). High-level mupirocin resistance in Staphylococcus aureus: evidence for two distinct isoleucyl-tRNA synthetases. Antimicrobial Agents and Chemotherapy 37 , 328.[Abstract]
11 . Snell, J. J., Brown, D. F., Perry, S. F. & George, R. (1993). Antimicrobial susceptibility testing of enterococci: results of a survey conducted by the United Kingdom National External Quality Assessment Scheme for Microbiology. Journal of Antimicrobial Chemotherapy32 , 40111.[Abstract]
12 . National Committee for Clinical Laboratory Standards. (1994). Performance Standards for Antimicrobial Susceptibility TestingFifth Informational Supplement M100-S5 . NCCLS, Villanova, PA.
13 . Stratton, C. W., Gelfand, M. S., Gerberding, J. L. & Chambers, H. F. (1990). Characterization of mechanisms of resistance to ß- lactam antibiotics in methicillin-resistant strains of Staphylococcus saprophyticus. Antimicrobial Agents and Chemotherapy 34, 17802.[ISI][Medline]
14 . Suzuki, E., Hiramatsu, K. & Yokota, T. (1992). Survey of methicillin-resistant clinical strains of coagulase-negative staphylococci for mecA gene distribution. Antimicrobial Agents and Chemotherapy 36, 42934.[Abstract]
15 . Hanaki, H., Kuwahara-Arai, K., Boyle-Vavra, S., Daum, R. S., Labischinski, H. & Hiramatsu, K. (1998). Activated cell-wall synthesis is associated with vancomycin resistance in methicillin- resistant Staphylococcus aureus clinical strains Mu3 and Mu50. Journal of Antimicrobial Chemotherapy 42, 199209.[Abstract]
16
.
Iwen, P. C., Kelly, D. M., Linder, J., Hinrichs, S. H., Dominguez, E. A., Rupp, M. E. et
al. (1997). Change in prevalence and antibiotic resistance of Enterococcus species
isolated from blood cultures over an 8-year period. Antimicrobial Agents and
Chemotherapy 41, 4945.
17
.
Tsakris, A., Pournaras, S. & Douboyas, J. (1997). Changes in antimicrobial
resistance of
enterococci isolated in Greece. Journal of Antimicrobial Chemotherapy 40, 7357.
18 . Appelbaum, P. C. (1992). Antimicrobial resistance in Streptococcus pneumoniae: an overview. Clinical Infectious Diseases 15, 7783.[ISI][Medline]
19 . Felmingham, D., Robbins, M. J., Dencer, C., Nathwani, A. & Gruneberg, R. N. (1996). Antimicrobial susceptibility of community-acquired bacterial lower respiratory tract pathogens. Journal of Antimicrobial Chemotherapy 38, 74751.[ISI][Medline]
20 . Schouten, M. A. & Hoogkamp-Korstanje, J. A. A. (1997). Comparative in-vitro activities of quinupristin-dalfopristin against Gram-positive bloodstream isolates. Journal of Antimicrobial Chemotherapy 40, 21319.[Abstract]
Received 20 April 1998; returned 17 June 1998; revised 22 July 1998; accepted 4 January 1999