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Epidemiological surveillance of Acinetobacter species
Journal
of Hospital
Infection
(1990)
Epidemiological
16, 319-329
surveillance species K. A. Wise
Department
of Medical
and F. A. Tosolini
Microbiology, Victoria,
Accepted
of Acinetobacter
Austin Australia
for publication
Hospital,
17 May
Heidelberg,
3084
1990
Summary:
Two hundred and sixty Acinetobacter isolates were recovered from 237 patients over a 2-year period; 1.56 isolates from 135 spinal cord injuries unit (SCIU) patients and 104 isolates from 102 patients in all the other hospital units. In SCIU patients, 133 isolates were recovered from the urine, 21 from wounds and aspirates, one from sputum and one from blood culture. In non-SCIU patients, 12 isolates were recovered from urine, 43 from wounds and aspirates, 48 from sputum and one from blood culture. Sixty-nine percent of isolates from SCIU patients showed resistance to gentamicin compared to 3% from non-SCIU patients. Gentamicin-resistant Acinetobacter anitratus was recovered from many environmental sites in the SCIU wards and from the hands of seven of 94 SCIU staff members tested. Serial rectal swabs were obtained from 79 newly-diagnosed SCIU patients. Ninety-two percent of those patients followed for up to 5 months acquired gentamicin-resistant Acinetobacter anitratus in their intestinal tract. API 2QNE profiles and antibiograms suggested that two distinct gentamicinresistant strains of A. anitratus had become endemic in the SCIU and that nosocomial transmission was a frequent occurrence. Keywords: infection.
Acinetobacter
surveillance;
gentamicin
resistance;
nosocomial
Introduction
Acinetobacter species are emerging as common nosocomial pathogens, especially involving the urinary and respiratory tracts and wound infections (Glew, Moellering & Kunz, 1977; Bergogne-B&&in & Joly-Guillou, 1985; Bergogne-Berizin, Joly-Guillou & Vieu, 1987). The organisms can be isolated from many environmental sources, including the hospital environment. They also form part of the normal flora and may colonize the intestinal tract of hospital patients. These organisms grow readily on routine bacteriological media, but are biochemically relatively inert, therefore it may be difficult to know whether different isolates are related epidemiologically. Many methods have been Correspondence Wollongong
2500,
019S-6701/90/080319+11
to: Dr K. A. Wise, Department New South Wales, Australia.
of Microbiology,
Wollongong
Hospital, 0 1990 The Hosptal
$03.00/O
319
Crown Infection
Street, Soaety
320
K. A. Wise and F. A. Tosolini
used to study the epidemiology of Acinetobacter including: antibiograms combined with selected biochemical traits (French et al., 1980; Holton, 1982; Allen & Green, 1987; Gerner-Smidt, 1987); combinations of polyacrylamide gel electrophoresis, plasmid analysis, antibiogram and biochemical reactions (Mortensen et al., 1987; Alexander et al., 1988; Vila, Almela & Jiminez de Anta, 1989); API 20NE profiles and antibiograms (Towner & Chopade, 1987); bacteriocin typing (Andrews, 1986) and serotyping (Das & Ayliffe, 1984). Some of these test methods are time-consuming, difficult to perform, expensive and not usually available in routine diagnostic laboratories. The availability of a test that is reproducible and easy to perform would allow epidemiological studies to be readily available when required. The API 20NE system fulfils these criteria (Towner & Chopade, 1987). The API 20NE identification system consists of a series of wells which contain different substrates, including 12 carbohydrate assimilation reaction wells. The pattern of reactions corresponds to a numerical figure which can be used to differentiate between isolates. We used this system, together with antibiograms, to perform an epidemiological investigation of isolates of Acinetobacter encountered at the Austin Hospital. Patients
and
methods
Clinical isolates Two hundred and sixty consecutive clinical isolates of Acinetobucter species were examined over a 2-year period from July 1987 to June 1989. One hundred and fifty-six isolates were recovered from 135 patients in the Spinal Cord Injuries Unit (SCIU), and 104 isolates were recovered from 102 patients from all other units and departments of the hospital. Environmental sampling One hundred and seventy-three environmental swabs were performed in the SCIU wards, Physiotherapy and Occupational Therapy departments. Wet areas (shower floors, shower chairs, shower tables, sinks, mops, buckets, soap and pan rooms) and dry areas (beds, bedding, curtains, furniture, drug trolleys, stock cupboards, equipment and linen storage areas, physiotherapy and occupational therapy equipment and treatment areas) were swabbed for the presence of gentamicin-resistant (GR) Acinetobacter strains. StafS screening Ninety-four staff members who had direct contact with patients from the SCIU (including nursing, medical, paramedical and domestic staff) were screened for the presence of GR Acinetobacter. All staff washed their hands before being swabbed. Cotton swabs were moistened with sterile saline and rubbed over the hands, between the fingers and under rings and watches.
Acinetobacter
surveillance
321
Rectal swabs A total of 259 rectal swabs was screened for CR Acinetobacter species from 79 consecutive newly admitted SCIU patients. All patients had at least one swab performed. Swabs were taken routinely on admission. Thereafter, where possible, weekly swabs were performed for the first month after admission, fortnightly for the next month, then monthly for as long as the patient remained in hospital. An additional 53 rectal swabs were performed on 31 previously-treated SCIU patients who were re-admitted for other medical or surgical reasons. Microbiological investigations Urine, sputum and wound specimens were inoculated onto sheep blood and MacConkey agar and incubated overnight at 37°C in 5% CO, in air (except urine cultures, which were incubated in air only). Wound swabs were also inoculated onto Columbia base agar enriched with horse blood, menadione and haemin and incubated anaerobically at 37°C for up to 48 h. Aerobic and anaerobic blood cultures were processed as described by Isenberg et al. (1985). Any G ram-negative rods isolated were identified to species level as described below. Antibiotic sensitivities were determined by agar dilution. The method was based on that described in Washington (19851, with Australian modifications (Butcher et al., 1986). Isosensitest agar (Oxoid) was used as the base medium and the inoculum was approximately lo4 cfu of each isolate, Antibiotics tested (concentrations in mg 1-l) with this method were ampicillin (2,8,16), trimethoprim-sulphamethoxazole (1: 19), nitrofurantoin (641, nalidixic acid (16), cephalothin (8,32), cefotaxime (8,32), gentamicin (2,4,8), tobramycin (2,4,8), chloramphenicol (8), norfloxacin (16), ticarcillin (16,64) and ampicillin-sulbactam (8:4). Interpretative criteria were based on those of Butcher et al. (1986). Additional antibiotic sensitivity tests were performed on 63 GR isolates from SCIU patients using the Vitek automated microbial system (McDonnell Douglas Information Systems). Antibiotics tested were ceftriaxone, ceftazidime, cefuroxime, amikacin, ciprofloxacin, carbenicillin, piperacillin and mezlociliin. Interpretation of these results was provided automatically by the Vitek system after the test organism was identified to the system as Acinetobacter anitratus. The multipoint inoculation system was also used to inoculate a series of plates containing a range of biochemical substrates. The pattern of reactions obtained was used to provide a presumptive identification of the Acinetobacter species. The API 20NE system was then used to confirm the identity of these isolates. The numerical profile was recorded for subsequent epidemiological investigations. Environmental swabs, staff hand swabs and patient rectal swabs were inoculated onto MacConkey agar plates containing 8 mg 1-l of gentamicin and into nutrient broth with the same concentration of gentamicin and
K. A. Wise and F. A. Tosolini
322
incubated at 37°C in air for up to 48 h. After 18 h the nutrient broths were subcultured onto MacConkey agar plates containing 8 mg 1-i of gentamicin and incubated at 37°C in air for a further 48 h. Any GR organisms isolated were identified to species level by the agar system described above. The identification of any Acinetobacter species was confirmed in the API 20NE system and the numerical profile was recorded. Results
The source of the Acinetobacter species isolated from clinical specimens is shown in Table 1. Amongst the SCIU patients, 133 of the 156 isolates were from urine, 21 were from wounds or aspirates, and one each from sputum and blood cultures. This contrasts with the other hospital units, where only 12 of the 104 isolates were from urine, whereas 43 were from wounds or aspirates, 48 were from sputum and one was from blood cultures.
Spinal
unit patients
One hundred and eight GR Acinetobacter isolates were recovered from 105 SCIU patients over the 2-year period of this study. New isolates occurred in each month of the study, ranging from 2 to 10 per month but with no obvious trends over time, suggesting that these organisms were endemic in the SCIU. As shown in Table II, 107 of the 108 isolates had an API 20NE profile of 0001073, and one was a biological variant which did not assimilate citrate, thus giving the profile 0001072. The API 20NE was repeated for 26 of these isolates. All of these isolates showed the same profile on repeat testing.
Table
I.
Sources
of
Acinetobacter sensitivity (GS)
species in clinical or resistance (GR) No.
Source
(no.
Urine GR GS
(145)
Wounds/aspirates
unit
in
relation
from Other
units
98 35
1 11
vi
2 41
Total
99 46
(49)
0 iE Blood
grouped
(64)
E Sputum
of patients
Spinal
isolates)
specimens, to gentamicin
1 culture
Total
(260)
0 48
0 49
(2)
0
0
0
1
1
2
1.56
104
260
to
Acinetobacter
Table II.
API
Acinetobacter
ZONE
profiles
of
Acinetobacter
species
isolates
in the Spinal
Cord
Injuries
Unit
API 20NE profile
No. of isolates
0001073 0001072
107 1
Gentamicin-resistant: A. anitratus
323
surveillance
(108)
Gentamicin-sensitive: A. anitratus
A. lwofi
(45)
0001073 0041073 0041473 0001071 0041071 0041471 4001073 0001072 0001473 4000073
(3)
24 6 4 i 2 2 1 :
0000032 000005 1 0000071
Two distinct antibiograms were found, as shown in Table II I. One group of isolates (24%) was sensitive to ticarcillin and ampicillin-sulbactam, and the other group (76%) was resistant to these agents. The patterns of sensitivity were consistent except for cefotaxime. This antibiotic was tested in our agar dilution system at concentrations of 8 mg 1-i and 32 mg I-‘. Sixty isolates were inhibited by the lower concentration while 48 isolates grew in the presence of the lower concentration but were inhibited by the higher concentration. There was no correlation with the pattern seen with ticarcillin and ampicillin-sulbactam. It is most likely that these isolates have Table
I II.
Antibiograms of 108 GR Acinetobacter anitratus specimens from patients in the Spinal Cord
Antimicrobial* Ampicillin Cotrimoxazole Nitrofurantoin Nalidixic acid Cephalothin Cefotaxime Gentamicin Tobramycin Chloramphenicol Norfloxacin Ticarcillin Ampicillin/Sulbactam * Carbenicillin, piperacillin and mezlocillin smpicillin/sulbactam in that all strains tested ** See Results section.
No.
susceptible
(%)
0 (0) 0 (0) 0 (0) 0 (0) -**0 (0) 0 (0) 108 0 0 26 26
(100) (0) (0) (24) (24)
sensitivity followed were either sensitive
isolates recovered Injuries Unit No.
from
resistant 108 108 108 108 108 -** 108 0 108 108 82 82
clinical
(%)
(100) (100) (100) (100) (100) (100) (0) (100) (100) (76) (76)
a similar pattern to ticarcillin or resistant to all agents.
and
K. A. Wise and F. A. Tosolini
324 Table Acinetobacter
IV.
API ZONE profiles in units other than the Spinal Cord Injuries
species
API 20NE
Gentamicin-resistant: A. anitratus (3) Gentamicin-sensitive: A. anitratus (73)
A. lwofi (19)
A. haemolyticus-alcaligenes
profile
No. of isolates
0001073
3
0001073 0041073 0001071 0041473 0001072 0040073 0041071 0000073 0041453 0040473 0001053
36 10 6 6 5 3 2 2 1
0000053 0000050 0000072 0200051 0000010 0000032 0000042 000005 1 0000070 0200050 0200071 (9)
Unit
0010053 0010050 0010051 0000053 4000050
:
: : ; 2 1 1
an MIC very close to 8 mg 1-l and that inoculum effects resulted in the observed variance. Sixty-three of these GR isolates also had further sensitivities performed using the Vitek system. All isolates were sensitive to ceftazidime and amikacin, and resistant to cefuroxime and ciprofloxacin. Carbenicillin, paralleled ticarcillin and piperacillin and mezlocillin sensitivities ampicillin-sulbactam: that is, an individual isolate was either sensitive or resistant to all of these agents. Sixty (95%) of the isolates tested were sensitive to ceftriaxone, while three (So/o) were intermediate. We suspect that, as with cefotaxime, the MIC was close to the cut-off point for this antibiotic leading to this result. The API 20NE profile (with the exception of one variant) combined with a consistent and specific antibiogram pattern suggested that two distinct GR strains of A. anitratus were endemic in the SCIU and that nosocomial transmission was a common occurrence. We also recovered 48 gentamicin-sensitive (GS) isolates from patients in
Acinetobacter
surveillance
325
the SCIU. Forty-five of these were A. anitratus and three were A. Zwoji. A range of API 20NE profiles (Table II) and antibiograms was found (data not shown). Although profile number 0001073 was also the most common in these isolates, there was significant variation in antibiotic sensitivity patterns. A small number of isolates recovered over the 2-year study period had similar antibiogram patterns, but there was no epidemiological evidence to suggest that nosocomial transmission of these isolates had occurred. Other patients There were 104 Acinetobacter isolates recovered from patients in units other than the SCIU (Tables I and IV). Three isolates were resistant to gentamicin and were identified as A. anitratus. Two GR strains had identical API 20NE profiles and antibiograms to the SCIU isolates. Both patients had been treated by members of the SCIU team, suggesting nosocomial spread to these patients. The other patient had a GR strain with a completely different antibiogram. The 101 GS isolates, comprising 73 A. anitratus isolates, 19 A. Zwo$i isolates and 9 A. haemolyticus-alcaligenes isolates, had various API 20NE profiles and antibiograms. The commonest profile was again 0001073, seen in 36 isolates. There were proportionately more A. Zwo#i and A. haemolyticus-alcaligenes isolates found in this group. A small number of Acinetobacter isolates recovered from the sputum of patients in the Intensive Care Unit (ICU) showed API 20NE profiles and antibiogram patterns similar to each other suggesting that nosocomial acquisition may have occurred at times here, but there was no sustained series of cases which required active infection control intervention. Environmental sampling Twenty-four GR Acinetobacter isolates were recovered from the 173 environmental swabs. All of these organisms had the same API 20NE profile and one of the two antibiogram patterns shown by the GR isolates recovered from the clinical specimens. These organisms were isolated from both wet and dry areas, including shower trolleys and seats, toilet seats and floors, pan room sinks and floors, corridor floors, clean equipment trolleys, bedding of unoccupied beds, stored clean pillows, shared pillows, balance support rails and floor mats in rehabilitation areas. Staff screening Seven staff members had GR Acinetobacter species recovered from swabs of their hands. All of these isolates had the same API 20NE profile and one of the two antibiogram patterns shown by the GR isolates recovered from clinical specimens taken from the SCIU patients. Rectal swabs Thirty-three of 79 newly admitted
SCIU
patients became colonized in the
K. A. Wise and F. A. Tosolini
326 Table
Length (months)
Cumulative acquisition of gentamicin-resistant Acinetobacter species in the intestinal tract of newly admitted Spinal Cord Injuries Unit patients
V.
of time
On admission 1 month 2 months 3 months 4 months 5 months
followed
No.
of patients
studied
No.
colonized
Acinetobacter 24 55 47 41 36
0 9 22 26 30 33
with species
GR (%)
(15) (40) (55) (73) (92)
bowel with GR Acinetobacter species. These 33 patients acquired their GR Acinetobacter from 10 days to 5 months after admission (mean 2.1 months, median 1.0 month). Acinetobacter sp. were not isolated from the swabs from any of these patients on admission, indicating nosocomial acquisition of these organisms. The cumulative acquisition of Acinetobacter isolates is shown in Table V. The antibiogram pattern and API 20NE profile confirmed that the strains were of the same two types isolated from clinical specimens. Most colonized patients continued to excrete the organism during follow-up testing. Nine (29%) of the 31 previously treated SCIU patients readmitted with other conditions were also found to be colonized with GR Acinetobacter species. All of these Acinetobacter isolates were of the same two types as those found among the clinical isolates. Discussion
The taxonomy of the genus Acinetobacter has recently been investigated by Bouvet & Grimont (1986). Using deoxyribonucleic acid hybridization methods they identified 12 distinct hybridization groups. As a result of this work, amendments to previously described species and additional species have been proposed. In this study we used the API 20NE to identify isolates and to provide the profile number on which our epidemiological studies were partly based. API 20NE identifies three Acinetobacter species: A. anitratus, A. lwofi and A. haemolyticus-alcaligenes. These species can be sub-divided into 209 biotypes based on the pattern of biochemical reactions obtained. The Vitek system was used for extra sensitivities on GR A. anitratus isolates. A. anitratw is also the name provided in the Vitek software for keying in the identification of the organism being tested. Therefore, in this paper, we elected to use the names still currently provided for the genus Acinetobacter by these commercial manufacturers. The aim of the study was to examine whether the API 20NE profile combined with the antibiogram was useful for the investigation of the epidemiology of Acinetobacter species. We found 31 different biotypes amongst the 260 clinical isolates of Acinetobacter species tested (Tables II
Acinetobacter
surveillance
327
and IV). The predominant biotype was A. anitratus profile number 0001073, both in the SCIU (Table II) and in the other hospital units (Table IV). Most isolates of this biotype (65%) were resistant to gentamicin (82% in the SCIU compared with 8% in the other hospital units). Towner & Chopade (1987) studied 122 clinical Acinetobacter isolates with this system and also found 31 different biotypes. Two biotypes predominated in their series, 18 GR A. anitratus isolates with profile number 4041473, and 19 GS A. Zwofi isolates with profile number 0000052. They noted that the A. anitrahs with profile number 4041473 had a distinctive antibiogram which included resistance to gentamicin, was endemic in the hospitals in the area and was associated with nosocomial infections in those hospitals. In our study, all of the 108 GR A. anitratus isolates recovered from SCIU patients showed an API 20NE profile of 0001073 (except for one biochemical variant shown in Table II), and all had one of two characteristic antibiograms (Table III). Th is suggested that two distinct strains had become endemic in the SCIU and appeared to have spread. In contrast, although some of the GS Acinetobacter isolates in our study had similar API 20NE profiles and antibiotic sensitivity patterns, there was no obvious epidemiological connection over the two year study period and no evidence of sustained nosocomial transmission of any of these strains. Swabs taken from the environment and from staff members’ hands indicated widespread contamination with two GR strains of A. anitratus. Similarly, rectal swabbing of SCIU patients showed that intestinal carriage of these same two strains was common and that they were carried in large numbers in affected patients, resulting in a large reservoir for the potential dissemination of these organisms. The frequency of intestinal carriage of these strains in newly-admitted SCIU patients increased with time (Table V), and previously treated SCIU patients re-admitted for other reasons commonly harboured GR Acinetobacter species in their intestinal tract. It is possible that intestinal carriage in staff members may also be a potential reservoir for these organisms. Shlaes et al. (1983) have reported the epidemiology of patient colonization with GR Gram-negative organisms in spinal injuries patients. They found that Acinetobacter species were more likely to colonize the bowel than other sites, and that nosocomial acquisition of GR organisms was directly related to the length of hospital stay and was not related to antibiotic use in general nor aminoglycoside use in particular. We believe that the figures shown in Table V and the fact that most of the newly diagnosed SCIU patients did not receive aminoglycoside antibiotics (data not shown) support these findings. The most likely mode of spread of the GR Acinetobacter strains in the SCIU is via the hands of staff and patients. This is consistent with the known reservoirs of these organisms in the SCIU, namely the intestinal and urinary tracts of affected patients. Other groups have found colonization of the hands of staff with Acinetobacter species to be common, though usually
328
K. A. Wise and F. A. Tosolini
transient, when nosocomial outbreaks were investigated (French et al., 1980; Holton, 1982; Allen & Green, 1987; Gerner-Smidt, 1987). French et al. (1980) also found that elimination of environmental contamination did not alter the course of an Acinetobacter outbreak that they investigated. Hartstein et al. (1988) reported an outbreak of Acinetobacter respiratory tract infections in patients in intensive care that was not responsive to barrier isolation methods and improved hand washing, but was promptly controlled by pasteurization of reusable ventilator circuits and resuscitation bags. More recently, the possibility of airborne spread of Acinetobacter has been raised (Allen & Green, 1987; Gerner-Smidt, 1987). We, and others (Fujita, Lilly & Ayliffe, 1982; Allen & Green, 1987), have been able to recover Acinetobacter species from ‘dry’ areas in the ward environment, a finding unusual for Gram-negative environmental organisms. This ability to survive drying could allow dissemination by airborne routes. The isolation of these organisms from ventilator tubing in intensive care patients (Gerner-Smidt, 1987; Hartstein et al., 1988) also indicates the possibility of airborne dissemination. Transmission by other routes, including inanimate objects, appears to be unlikely even though contamination of the environment may be high (French et al., 1980; Allen & Green, 1987; Gerner-Smidt, 1987). Strict infection control measures, highlighting hand-washing practices, were encouraged in both staff and patients in the SCIU. Ongoing surveillance has shown that the frequency of isolation of these GR Acinetobacter strains from clinical specimens has begun to fall in the SCIU at our hospital (data not shown). Eradication of these organisms from the SCIU will be difficult, mainly because of their affinity for the intestinal tract, so we have concentrated our efforts on interrupting their nosocomial transmission. Acinetobacter infections in patients from other hospital units were also relatively common, but it was reassuring to find no strong epidemiological evidence of nosocomial transmission. API 20NE profiles and antibiotic sensitivity patterns are not definitive epidemiological tools. However, in this study, the API 20NE profile and antibiogram were quite characteristic and we were confident that we were observing the spread of two endemic GR strains of A. anitratus in the SCIU in our hospital. The API 20NE provides a profile number that, when combined with the antibiogram, can be a rapid, simple and inexpensive way to investigate the epidemiology of these organisms. This combination could be used for the routine surveillance of all Acinetobacter isolates, and to assist in monitoring the spread of particular strains in the hospital environment, especially those isolated from specific units like the SCIU and ICU, and for more complete identification of Acinetobacter isolates when nosocomial transmission is suspected. The authors wish to thank Sr Wendy Barr, Sr Joan Booth, Dr Douglas Brown,and the spinal cord injuries unit for their assistance in the performance of this study.
the staff of
Acinetobacter
surveillance
329
References Alexander, M., Rahman, M., Taylor, M. & Noble, W. C. (1988). A study of the value of electrophoretic and other techniques for typing Acinetobacter calcoaceticus. Journal of Hospital Infection 12, 273-287. Allen, K. D. & Green, H. T. (1987). Hospital outbreak of multi-resistant Acinetobacter anitratus: an airborne mode of spread? Journal of Hospital Infection 9, 110-l 19. Andrews, H. J. (1986). Acinetobacter bacteriocin typing. Journal of Hospital Infection 7, 169-175., Bergogne-Berezin, E. & Joly-Guillou, M. L. (1985). An underestimated nosocomial pathogen, Acinetobacter calcoaceticus. Journal of Antimicrobial Chemotherapy 16, 535-538. Bergogne-Berezin, E., Joly-Guillou, M. L. & Vieu, J. F. (1987). Epidemiology of nosocomial infections due to Acinetobacter calcoaceticus. Journal of Hospital Infection 10, 105-l 13. Bouvet, P. J. M. & Grimont, P. A. D. (1986). Taxonomy of the genus Acinetobacter with the recognition of Acinetobacter baumannii sp. no”., Acinetobacter haemolyticus sp. no”., Acinetobacter johnsonii sp. no”., and Acinetobacter junii sp. nov. and amended descriptions of Acinetobacter calcoaceticus and Acinetobacter lwofl. InternationalJournal of Systematic Bacteriology 36, 228-240. Butcher, R., Franklin, C., Gilbert, G., Olden, D., Stockman, K. & Ward, P. (1986). Agar dilution susceptibility testing for bacteria that grow in air. Clinical Microbiology Update Programme, Number 27, July. Sydney, Australia: Australian Society for Microbiology (New South Wales Branch). French, G. L., Casewell, M. W., Roncoroni, A. J., Knight, S. & Phillips, I (1980). A hospital outbreak of antibiotic-resistant Acinetobacter anitratus: epidemiology and control. Journal of Hospital Infection 1, 125-l 3 1. Fujita, K., Lilly, H. A. & Ayliffe, G. A. J. (1982). Spread of resistant Gram-negative bacilli in a burns unit. Journal of Hospital Infection 3, 29-37. Gerner-Smidt, P. (1987). Endemic occurrence of Acinetobacter calcoaceticus biovar anitratus in an intensive care unit. Journal of Hospital Infection 10, 265-272. Glew, R. H., Moellering, R. C. & Kunz, L. J. (1977). Infections with Acinetobacter calcoaceticus (Herellea vaginicola): Clinical and laboratory studies. Medicine 56, 79-97. Hartstein, A. I., Rashad, A. L., Liebler, J. M., Actis, L. A., Freeman, J., Rourke, J. W., Stibolt, T. B., Tolmasky, M. E., Ellis, G. R. & C rosa, J. H. (1988). Multiple intensive care unit outbreak of Acinetobacter calcoaceticus subspecies anitratus respiratory infection and colonization associated with contaminated, reusable ventilator circuits and resuscitation bags. American Journal of Medicine 85, 624-63 1. Holton, J. (1982). A report of a further hospital outbreak caused by a multi-resistant Acinetobacter anitratus. Journal of Hospital Infection 3, 305-309. Isenberg, H. D., Washington, J. A., Balows, A. & Sonnenwirth, A. C. (1985). Collection, handling, and processing of specimens. In Manual of Clinical Microbiology, 4th edn (Lennette, E. H., Balows, A., Hausler, W. J. & Shadomy, H. J., Eds.), pp. 73-98. Washington, D.C: American Society for Microbiology. Mortensen, J. E., LaRocco, M. T., Steiner, B. & Ribner, B. (1987). Protein fingerprinting for the determination of relatedness in Acinetobacter calcoaceticus subspecies anitratus isolated from patients in a surgical intensive care unit. Infection Control t, 512-515. Shlaes, D. M., Currie, C. A., Rotter, G., Eanes, M. & Floyd, R. (1983). Epidemiology of gentamicin-resistant, Gram-negative bacillary colonization in a spinal cord injury unit. rournal of Clinical Microbiology 18, 227-235. Towner, K. J. & Chopade, B. A. (1987). Biotyping of Acinetobacter calcoaceticus using the API 20NE system. Journal of Hospital Infection 10, 145-l 5 1. Vila, J., Almela, M. & Jimenez de Anta, M. T. (1989). Laboratory investigation of hospital outbreak caused by two different multiresistant Acinetobacter calcoaceticus subsp. anitratus strains. Journal of Clinical Microbiology 27, 1086-1089. Washington, J. A. (1985). Susceptibility tests: agar dilution. In Manual of Clinical Microbiology, 4th edn (Lennette, E. H., Balows, A., Hausler, W. J. & Shadomy, H. J., Eds.), pp. 967-971. Washington, D.C: American Society for Microbiology.
of Hospital
Infection
(1990)
Epidemiological
16, 319-329
surveillance species K. A. Wise
Department
of Medical
and F. A. Tosolini
Microbiology, Victoria,
Accepted
of Acinetobacter
Austin Australia
for publication
Hospital,
17 May
Heidelberg,
3084
1990
Summary:
Two hundred and sixty Acinetobacter isolates were recovered from 237 patients over a 2-year period; 1.56 isolates from 135 spinal cord injuries unit (SCIU) patients and 104 isolates from 102 patients in all the other hospital units. In SCIU patients, 133 isolates were recovered from the urine, 21 from wounds and aspirates, one from sputum and one from blood culture. In non-SCIU patients, 12 isolates were recovered from urine, 43 from wounds and aspirates, 48 from sputum and one from blood culture. Sixty-nine percent of isolates from SCIU patients showed resistance to gentamicin compared to 3% from non-SCIU patients. Gentamicin-resistant Acinetobacter anitratus was recovered from many environmental sites in the SCIU wards and from the hands of seven of 94 SCIU staff members tested. Serial rectal swabs were obtained from 79 newly-diagnosed SCIU patients. Ninety-two percent of those patients followed for up to 5 months acquired gentamicin-resistant Acinetobacter anitratus in their intestinal tract. API 2QNE profiles and antibiograms suggested that two distinct gentamicinresistant strains of A. anitratus had become endemic in the SCIU and that nosocomial transmission was a frequent occurrence. Keywords: infection.
Acinetobacter
surveillance;
gentamicin
resistance;
nosocomial
Introduction
Acinetobacter species are emerging as common nosocomial pathogens, especially involving the urinary and respiratory tracts and wound infections (Glew, Moellering & Kunz, 1977; Bergogne-B&&in & Joly-Guillou, 1985; Bergogne-Berizin, Joly-Guillou & Vieu, 1987). The organisms can be isolated from many environmental sources, including the hospital environment. They also form part of the normal flora and may colonize the intestinal tract of hospital patients. These organisms grow readily on routine bacteriological media, but are biochemically relatively inert, therefore it may be difficult to know whether different isolates are related epidemiologically. Many methods have been Correspondence Wollongong
2500,
019S-6701/90/080319+11
to: Dr K. A. Wise, Department New South Wales, Australia.
of Microbiology,
Wollongong
Hospital, 0 1990 The Hosptal
$03.00/O
319
Crown Infection
Street, Soaety
320
K. A. Wise and F. A. Tosolini
used to study the epidemiology of Acinetobacter including: antibiograms combined with selected biochemical traits (French et al., 1980; Holton, 1982; Allen & Green, 1987; Gerner-Smidt, 1987); combinations of polyacrylamide gel electrophoresis, plasmid analysis, antibiogram and biochemical reactions (Mortensen et al., 1987; Alexander et al., 1988; Vila, Almela & Jiminez de Anta, 1989); API 20NE profiles and antibiograms (Towner & Chopade, 1987); bacteriocin typing (Andrews, 1986) and serotyping (Das & Ayliffe, 1984). Some of these test methods are time-consuming, difficult to perform, expensive and not usually available in routine diagnostic laboratories. The availability of a test that is reproducible and easy to perform would allow epidemiological studies to be readily available when required. The API 20NE system fulfils these criteria (Towner & Chopade, 1987). The API 20NE identification system consists of a series of wells which contain different substrates, including 12 carbohydrate assimilation reaction wells. The pattern of reactions corresponds to a numerical figure which can be used to differentiate between isolates. We used this system, together with antibiograms, to perform an epidemiological investigation of isolates of Acinetobacter encountered at the Austin Hospital. Patients
and
methods
Clinical isolates Two hundred and sixty consecutive clinical isolates of Acinetobucter species were examined over a 2-year period from July 1987 to June 1989. One hundred and fifty-six isolates were recovered from 135 patients in the Spinal Cord Injuries Unit (SCIU), and 104 isolates were recovered from 102 patients from all other units and departments of the hospital. Environmental sampling One hundred and seventy-three environmental swabs were performed in the SCIU wards, Physiotherapy and Occupational Therapy departments. Wet areas (shower floors, shower chairs, shower tables, sinks, mops, buckets, soap and pan rooms) and dry areas (beds, bedding, curtains, furniture, drug trolleys, stock cupboards, equipment and linen storage areas, physiotherapy and occupational therapy equipment and treatment areas) were swabbed for the presence of gentamicin-resistant (GR) Acinetobacter strains. StafS screening Ninety-four staff members who had direct contact with patients from the SCIU (including nursing, medical, paramedical and domestic staff) were screened for the presence of GR Acinetobacter. All staff washed their hands before being swabbed. Cotton swabs were moistened with sterile saline and rubbed over the hands, between the fingers and under rings and watches.
Acinetobacter
surveillance
321
Rectal swabs A total of 259 rectal swabs was screened for CR Acinetobacter species from 79 consecutive newly admitted SCIU patients. All patients had at least one swab performed. Swabs were taken routinely on admission. Thereafter, where possible, weekly swabs were performed for the first month after admission, fortnightly for the next month, then monthly for as long as the patient remained in hospital. An additional 53 rectal swabs were performed on 31 previously-treated SCIU patients who were re-admitted for other medical or surgical reasons. Microbiological investigations Urine, sputum and wound specimens were inoculated onto sheep blood and MacConkey agar and incubated overnight at 37°C in 5% CO, in air (except urine cultures, which were incubated in air only). Wound swabs were also inoculated onto Columbia base agar enriched with horse blood, menadione and haemin and incubated anaerobically at 37°C for up to 48 h. Aerobic and anaerobic blood cultures were processed as described by Isenberg et al. (1985). Any G ram-negative rods isolated were identified to species level as described below. Antibiotic sensitivities were determined by agar dilution. The method was based on that described in Washington (19851, with Australian modifications (Butcher et al., 1986). Isosensitest agar (Oxoid) was used as the base medium and the inoculum was approximately lo4 cfu of each isolate, Antibiotics tested (concentrations in mg 1-l) with this method were ampicillin (2,8,16), trimethoprim-sulphamethoxazole (1: 19), nitrofurantoin (641, nalidixic acid (16), cephalothin (8,32), cefotaxime (8,32), gentamicin (2,4,8), tobramycin (2,4,8), chloramphenicol (8), norfloxacin (16), ticarcillin (16,64) and ampicillin-sulbactam (8:4). Interpretative criteria were based on those of Butcher et al. (1986). Additional antibiotic sensitivity tests were performed on 63 GR isolates from SCIU patients using the Vitek automated microbial system (McDonnell Douglas Information Systems). Antibiotics tested were ceftriaxone, ceftazidime, cefuroxime, amikacin, ciprofloxacin, carbenicillin, piperacillin and mezlociliin. Interpretation of these results was provided automatically by the Vitek system after the test organism was identified to the system as Acinetobacter anitratus. The multipoint inoculation system was also used to inoculate a series of plates containing a range of biochemical substrates. The pattern of reactions obtained was used to provide a presumptive identification of the Acinetobacter species. The API 20NE system was then used to confirm the identity of these isolates. The numerical profile was recorded for subsequent epidemiological investigations. Environmental swabs, staff hand swabs and patient rectal swabs were inoculated onto MacConkey agar plates containing 8 mg 1-l of gentamicin and into nutrient broth with the same concentration of gentamicin and
K. A. Wise and F. A. Tosolini
322
incubated at 37°C in air for up to 48 h. After 18 h the nutrient broths were subcultured onto MacConkey agar plates containing 8 mg 1-i of gentamicin and incubated at 37°C in air for a further 48 h. Any GR organisms isolated were identified to species level by the agar system described above. The identification of any Acinetobacter species was confirmed in the API 20NE system and the numerical profile was recorded. Results
The source of the Acinetobacter species isolated from clinical specimens is shown in Table 1. Amongst the SCIU patients, 133 of the 156 isolates were from urine, 21 were from wounds or aspirates, and one each from sputum and blood cultures. This contrasts with the other hospital units, where only 12 of the 104 isolates were from urine, whereas 43 were from wounds or aspirates, 48 were from sputum and one was from blood cultures.
Spinal
unit patients
One hundred and eight GR Acinetobacter isolates were recovered from 105 SCIU patients over the 2-year period of this study. New isolates occurred in each month of the study, ranging from 2 to 10 per month but with no obvious trends over time, suggesting that these organisms were endemic in the SCIU. As shown in Table II, 107 of the 108 isolates had an API 20NE profile of 0001073, and one was a biological variant which did not assimilate citrate, thus giving the profile 0001072. The API 20NE was repeated for 26 of these isolates. All of these isolates showed the same profile on repeat testing.
Table
I.
Sources
of
Acinetobacter sensitivity (GS)
species in clinical or resistance (GR) No.
Source
(no.
Urine GR GS
(145)
Wounds/aspirates
unit
in
relation
from Other
units
98 35
1 11
vi
2 41
Total
99 46
(49)
0 iE Blood
grouped
(64)
E Sputum
of patients
Spinal
isolates)
specimens, to gentamicin
1 culture
Total
(260)
0 48
0 49
(2)
0
0
0
1
1
2
1.56
104
260
to
Acinetobacter
Table II.
API
Acinetobacter
ZONE
profiles
of
Acinetobacter
species
isolates
in the Spinal
Cord
Injuries
Unit
API 20NE profile
No. of isolates
0001073 0001072
107 1
Gentamicin-resistant: A. anitratus
323
surveillance
(108)
Gentamicin-sensitive: A. anitratus
A. lwofi
(45)
0001073 0041073 0041473 0001071 0041071 0041471 4001073 0001072 0001473 4000073
(3)
24 6 4 i 2 2 1 :
0000032 000005 1 0000071
Two distinct antibiograms were found, as shown in Table II I. One group of isolates (24%) was sensitive to ticarcillin and ampicillin-sulbactam, and the other group (76%) was resistant to these agents. The patterns of sensitivity were consistent except for cefotaxime. This antibiotic was tested in our agar dilution system at concentrations of 8 mg 1-i and 32 mg I-‘. Sixty isolates were inhibited by the lower concentration while 48 isolates grew in the presence of the lower concentration but were inhibited by the higher concentration. There was no correlation with the pattern seen with ticarcillin and ampicillin-sulbactam. It is most likely that these isolates have Table
I II.
Antibiograms of 108 GR Acinetobacter anitratus specimens from patients in the Spinal Cord
Antimicrobial* Ampicillin Cotrimoxazole Nitrofurantoin Nalidixic acid Cephalothin Cefotaxime Gentamicin Tobramycin Chloramphenicol Norfloxacin Ticarcillin Ampicillin/Sulbactam * Carbenicillin, piperacillin and mezlocillin smpicillin/sulbactam in that all strains tested ** See Results section.
No.
susceptible
(%)
0 (0) 0 (0) 0 (0) 0 (0) -**0 (0) 0 (0) 108 0 0 26 26
(100) (0) (0) (24) (24)
sensitivity followed were either sensitive
isolates recovered Injuries Unit No.
from
resistant 108 108 108 108 108 -** 108 0 108 108 82 82
clinical
(%)
(100) (100) (100) (100) (100) (100) (0) (100) (100) (76) (76)
a similar pattern to ticarcillin or resistant to all agents.
and
K. A. Wise and F. A. Tosolini
324 Table Acinetobacter
IV.
API ZONE profiles in units other than the Spinal Cord Injuries
species
API 20NE
Gentamicin-resistant: A. anitratus (3) Gentamicin-sensitive: A. anitratus (73)
A. lwofi (19)
A. haemolyticus-alcaligenes
profile
No. of isolates
0001073
3
0001073 0041073 0001071 0041473 0001072 0040073 0041071 0000073 0041453 0040473 0001053
36 10 6 6 5 3 2 2 1
0000053 0000050 0000072 0200051 0000010 0000032 0000042 000005 1 0000070 0200050 0200071 (9)
Unit
0010053 0010050 0010051 0000053 4000050
:
: : ; 2 1 1
an MIC very close to 8 mg 1-l and that inoculum effects resulted in the observed variance. Sixty-three of these GR isolates also had further sensitivities performed using the Vitek system. All isolates were sensitive to ceftazidime and amikacin, and resistant to cefuroxime and ciprofloxacin. Carbenicillin, paralleled ticarcillin and piperacillin and mezlocillin sensitivities ampicillin-sulbactam: that is, an individual isolate was either sensitive or resistant to all of these agents. Sixty (95%) of the isolates tested were sensitive to ceftriaxone, while three (So/o) were intermediate. We suspect that, as with cefotaxime, the MIC was close to the cut-off point for this antibiotic leading to this result. The API 20NE profile (with the exception of one variant) combined with a consistent and specific antibiogram pattern suggested that two distinct GR strains of A. anitratus were endemic in the SCIU and that nosocomial transmission was a common occurrence. We also recovered 48 gentamicin-sensitive (GS) isolates from patients in
Acinetobacter
surveillance
325
the SCIU. Forty-five of these were A. anitratus and three were A. Zwoji. A range of API 20NE profiles (Table II) and antibiograms was found (data not shown). Although profile number 0001073 was also the most common in these isolates, there was significant variation in antibiotic sensitivity patterns. A small number of isolates recovered over the 2-year study period had similar antibiogram patterns, but there was no epidemiological evidence to suggest that nosocomial transmission of these isolates had occurred. Other patients There were 104 Acinetobacter isolates recovered from patients in units other than the SCIU (Tables I and IV). Three isolates were resistant to gentamicin and were identified as A. anitratus. Two GR strains had identical API 20NE profiles and antibiograms to the SCIU isolates. Both patients had been treated by members of the SCIU team, suggesting nosocomial spread to these patients. The other patient had a GR strain with a completely different antibiogram. The 101 GS isolates, comprising 73 A. anitratus isolates, 19 A. Zwo$i isolates and 9 A. haemolyticus-alcaligenes isolates, had various API 20NE profiles and antibiograms. The commonest profile was again 0001073, seen in 36 isolates. There were proportionately more A. Zwo#i and A. haemolyticus-alcaligenes isolates found in this group. A small number of Acinetobacter isolates recovered from the sputum of patients in the Intensive Care Unit (ICU) showed API 20NE profiles and antibiogram patterns similar to each other suggesting that nosocomial acquisition may have occurred at times here, but there was no sustained series of cases which required active infection control intervention. Environmental sampling Twenty-four GR Acinetobacter isolates were recovered from the 173 environmental swabs. All of these organisms had the same API 20NE profile and one of the two antibiogram patterns shown by the GR isolates recovered from the clinical specimens. These organisms were isolated from both wet and dry areas, including shower trolleys and seats, toilet seats and floors, pan room sinks and floors, corridor floors, clean equipment trolleys, bedding of unoccupied beds, stored clean pillows, shared pillows, balance support rails and floor mats in rehabilitation areas. Staff screening Seven staff members had GR Acinetobacter species recovered from swabs of their hands. All of these isolates had the same API 20NE profile and one of the two antibiogram patterns shown by the GR isolates recovered from clinical specimens taken from the SCIU patients. Rectal swabs Thirty-three of 79 newly admitted
SCIU
patients became colonized in the
K. A. Wise and F. A. Tosolini
326 Table
Length (months)
Cumulative acquisition of gentamicin-resistant Acinetobacter species in the intestinal tract of newly admitted Spinal Cord Injuries Unit patients
V.
of time
On admission 1 month 2 months 3 months 4 months 5 months
followed
No.
of patients
studied
No.
colonized
Acinetobacter 24 55 47 41 36
0 9 22 26 30 33
with species
GR (%)
(15) (40) (55) (73) (92)
bowel with GR Acinetobacter species. These 33 patients acquired their GR Acinetobacter from 10 days to 5 months after admission (mean 2.1 months, median 1.0 month). Acinetobacter sp. were not isolated from the swabs from any of these patients on admission, indicating nosocomial acquisition of these organisms. The cumulative acquisition of Acinetobacter isolates is shown in Table V. The antibiogram pattern and API 20NE profile confirmed that the strains were of the same two types isolated from clinical specimens. Most colonized patients continued to excrete the organism during follow-up testing. Nine (29%) of the 31 previously treated SCIU patients readmitted with other conditions were also found to be colonized with GR Acinetobacter species. All of these Acinetobacter isolates were of the same two types as those found among the clinical isolates. Discussion
The taxonomy of the genus Acinetobacter has recently been investigated by Bouvet & Grimont (1986). Using deoxyribonucleic acid hybridization methods they identified 12 distinct hybridization groups. As a result of this work, amendments to previously described species and additional species have been proposed. In this study we used the API 20NE to identify isolates and to provide the profile number on which our epidemiological studies were partly based. API 20NE identifies three Acinetobacter species: A. anitratus, A. lwofi and A. haemolyticus-alcaligenes. These species can be sub-divided into 209 biotypes based on the pattern of biochemical reactions obtained. The Vitek system was used for extra sensitivities on GR A. anitratus isolates. A. anitratw is also the name provided in the Vitek software for keying in the identification of the organism being tested. Therefore, in this paper, we elected to use the names still currently provided for the genus Acinetobacter by these commercial manufacturers. The aim of the study was to examine whether the API 20NE profile combined with the antibiogram was useful for the investigation of the epidemiology of Acinetobacter species. We found 31 different biotypes amongst the 260 clinical isolates of Acinetobacter species tested (Tables II
Acinetobacter
surveillance
327
and IV). The predominant biotype was A. anitratus profile number 0001073, both in the SCIU (Table II) and in the other hospital units (Table IV). Most isolates of this biotype (65%) were resistant to gentamicin (82% in the SCIU compared with 8% in the other hospital units). Towner & Chopade (1987) studied 122 clinical Acinetobacter isolates with this system and also found 31 different biotypes. Two biotypes predominated in their series, 18 GR A. anitratus isolates with profile number 4041473, and 19 GS A. Zwofi isolates with profile number 0000052. They noted that the A. anitrahs with profile number 4041473 had a distinctive antibiogram which included resistance to gentamicin, was endemic in the hospitals in the area and was associated with nosocomial infections in those hospitals. In our study, all of the 108 GR A. anitratus isolates recovered from SCIU patients showed an API 20NE profile of 0001073 (except for one biochemical variant shown in Table II), and all had one of two characteristic antibiograms (Table III). Th is suggested that two distinct strains had become endemic in the SCIU and appeared to have spread. In contrast, although some of the GS Acinetobacter isolates in our study had similar API 20NE profiles and antibiotic sensitivity patterns, there was no obvious epidemiological connection over the two year study period and no evidence of sustained nosocomial transmission of any of these strains. Swabs taken from the environment and from staff members’ hands indicated widespread contamination with two GR strains of A. anitratus. Similarly, rectal swabbing of SCIU patients showed that intestinal carriage of these same two strains was common and that they were carried in large numbers in affected patients, resulting in a large reservoir for the potential dissemination of these organisms. The frequency of intestinal carriage of these strains in newly-admitted SCIU patients increased with time (Table V), and previously treated SCIU patients re-admitted for other reasons commonly harboured GR Acinetobacter species in their intestinal tract. It is possible that intestinal carriage in staff members may also be a potential reservoir for these organisms. Shlaes et al. (1983) have reported the epidemiology of patient colonization with GR Gram-negative organisms in spinal injuries patients. They found that Acinetobacter species were more likely to colonize the bowel than other sites, and that nosocomial acquisition of GR organisms was directly related to the length of hospital stay and was not related to antibiotic use in general nor aminoglycoside use in particular. We believe that the figures shown in Table V and the fact that most of the newly diagnosed SCIU patients did not receive aminoglycoside antibiotics (data not shown) support these findings. The most likely mode of spread of the GR Acinetobacter strains in the SCIU is via the hands of staff and patients. This is consistent with the known reservoirs of these organisms in the SCIU, namely the intestinal and urinary tracts of affected patients. Other groups have found colonization of the hands of staff with Acinetobacter species to be common, though usually
328
K. A. Wise and F. A. Tosolini
transient, when nosocomial outbreaks were investigated (French et al., 1980; Holton, 1982; Allen & Green, 1987; Gerner-Smidt, 1987). French et al. (1980) also found that elimination of environmental contamination did not alter the course of an Acinetobacter outbreak that they investigated. Hartstein et al. (1988) reported an outbreak of Acinetobacter respiratory tract infections in patients in intensive care that was not responsive to barrier isolation methods and improved hand washing, but was promptly controlled by pasteurization of reusable ventilator circuits and resuscitation bags. More recently, the possibility of airborne spread of Acinetobacter has been raised (Allen & Green, 1987; Gerner-Smidt, 1987). We, and others (Fujita, Lilly & Ayliffe, 1982; Allen & Green, 1987), have been able to recover Acinetobacter species from ‘dry’ areas in the ward environment, a finding unusual for Gram-negative environmental organisms. This ability to survive drying could allow dissemination by airborne routes. The isolation of these organisms from ventilator tubing in intensive care patients (Gerner-Smidt, 1987; Hartstein et al., 1988) also indicates the possibility of airborne dissemination. Transmission by other routes, including inanimate objects, appears to be unlikely even though contamination of the environment may be high (French et al., 1980; Allen & Green, 1987; Gerner-Smidt, 1987). Strict infection control measures, highlighting hand-washing practices, were encouraged in both staff and patients in the SCIU. Ongoing surveillance has shown that the frequency of isolation of these GR Acinetobacter strains from clinical specimens has begun to fall in the SCIU at our hospital (data not shown). Eradication of these organisms from the SCIU will be difficult, mainly because of their affinity for the intestinal tract, so we have concentrated our efforts on interrupting their nosocomial transmission. Acinetobacter infections in patients from other hospital units were also relatively common, but it was reassuring to find no strong epidemiological evidence of nosocomial transmission. API 20NE profiles and antibiotic sensitivity patterns are not definitive epidemiological tools. However, in this study, the API 20NE profile and antibiogram were quite characteristic and we were confident that we were observing the spread of two endemic GR strains of A. anitratus in the SCIU in our hospital. The API 20NE provides a profile number that, when combined with the antibiogram, can be a rapid, simple and inexpensive way to investigate the epidemiology of these organisms. This combination could be used for the routine surveillance of all Acinetobacter isolates, and to assist in monitoring the spread of particular strains in the hospital environment, especially those isolated from specific units like the SCIU and ICU, and for more complete identification of Acinetobacter isolates when nosocomial transmission is suspected. The authors wish to thank Sr Wendy Barr, Sr Joan Booth, Dr Douglas Brown,and the spinal cord injuries unit for their assistance in the performance of this study.
the staff of
Acinetobacter
surveillance
329
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