MICROBIOLOGICAL MONITORING OF SEVERELY BURNED PATIENTS ADMITTED TO THE BURNS CENTRE IN BARI (ITALY) IN THE PERIOD 1989-92

Calvario A.**, Di Lonardo A.*, Larocca A.M.V.**, Parisi D.*, Montagna M.T**, Ressa M.* Silvestri A.**, Maggio G.*

* Cattedra di Chirurgia Plastica, Universit& degli Studi, Bari
** Istituto di Igiene, Universit6 degli Studi, Bari, Italy

SUMMARY. An infection-prevention programme conducted in severely burned patients admitted over a four-year period to the Bums Centre in the Bari Polyclinic (Italy) indicated that the aetiopathogenesis of infection was mainly due to Pseudomonas aeruginosa and Staphylococcus aureus. These species - sometimes multiresistant to various topical and systemic treatments - often aggravated prognosis in the patients and complicated any reconstructive treatment already in progress. The importance is underlined of a systematic programme of microbiological surveillance covering patients, staff and the environment in order to monitor the circulation of the various microbic species and thus to ensure more successful results in the various types of reconstructive treatment, including skin flaps.

The elevated incidence of infective complications in high-risk departments such as Burns Centres or Intensive Care Units can sometimes reach dangerous levels, also because of the patients' generally compromised physical state. Rapid colonization by often antibiotic-resistant bacterial flora can on the one hand create difficulties in the therapeutic approach and on the other compromise the success of some of the more modern reconstructive treatments, such as the use of expanded skin flaps (7, 12, 37). In this last case, bacterial and/or mycotic contamination often extremely difficult to control - is Probably responsible for the extreme variability in the percentage take of in vitro cultivated epithelium.
Extensively burned patients clearly require an environment with high levels of protective isolation. But this is not always easy to realize, if we consider some negative aspects of hospital conditions (poor level of awareness among staff, inadequate health training, insufficient microbiological tests, etc.). Hence the importance in a Burns Centre of a programme of surveillance, with aimed microbiological monitoring both of the patients and of the more significant spaces in the hospital environment. Such a programme is particularly useful for preparation of appropriate prevention protocols.
This article reports the results of a study conducted at the Bari Polyclinic Burns Centre in the years 1989-92, when periodic microbiological tests were performed on patients, medical and nursing staff, and the hospital environment, in order to identify the infective complications present.

In the period 1989-92 nineteen severely burned patients (age 2-45 years, TI3SA 40-70%) were selected for the study. They were all admitted within 48 h of the trauma and belonged to classes 3, 4 and 5 of the Roi Indices (3 1). On admission, and once a week thereafter, surface swabs and biopsies were taken of the burn areas, after cleansing with sterile physiological solution. The biopsies were performed using a disposable 4-mm diameter punch biopsy (3, 19, 40). The number of tests was limited to an average of two per patient in 1989-90. The number was subsequently correlated to the extent of the bums, i. e. an average of one for every 20% of burn surface, in order to achieve more complete results. The surface swabs and the bioptic material (after weighing and homogenization) were seeded in a number of culture media (agar-agar, Wurtz lactose agar, mannitol salt agar, blood agar, pseudo-sel agar), both directly and after enrichment in broth for 18-24 h at thermostat 37 'C. The strains were identified by morphological staining tests, biochemical tests (15) and, where indicated, serological tests using specific commercial antisera (6).
For mycetes research we used sterile gauzes placed on the burned skin areas and then deposited on the surface of a Sabouraud agar dish with 0.5 gm/1 of CAF; the strains were identified using techniques reported by Segretain et a]. (32). Quantitative, bacterial and mycotic research was performed on the bioptic material (after weighing and homogenization) by seeding 1 ral of dilutions in base 10 prepared in physiological solution respectively in Plate Count Agar and Sabouraud agar with incubation at thermostat 37 'C for 48 h and for mycetes successively at room temperature for 7-10 days.
In eight of the patients biopsies were performed twice: a dry biopsy was examined microbiologically and another, fixed in 10% formalin, was prepared for histological examination in order to measure the level of microbial contamination in the various tissue planes. In this latter case, after inclusion in paraffin, each fragment was used to make 20 5~t thick serial sections perpendicular to the skin surface (13, 14) which were stained with haernatoxylin-eosin, Gomori-Grocott, gram modified and methylene blue. The depth reached by microbial invasion (M / 0 400x and 100x) was differentiated by the values on the following scale (2 1):

In all, 67 out of the 88 biopsies taken from the patients were also subjected to histological examination.

Key: CA = Candida albicans; EC = Escherichia coli; KP = Klebsiella pneumoniae; Pl 2 = Pseudomonas aeruginosa serotype 12-, PMi = Proteus mirabilis; PMo = Proteus morganii; SA = Staphylococcus aureus; SF = Streptococcusfiaecalis  
* = culture research negative

The sampling was completed, when possible, by cultural examination of oropharyngeal swabs, catheters, blood, urine and faeces (in the last case for the presence of pathogenie enterobacteria).

The environmental investigations were conducted in the medication rooms at three different times of day (8 a.m., 12 a.m. after medication of the patients, and 2 p.m. after cleaning and disinfection of the rooms) and in the intensive care rooms (where the 12 o'clock check was suppressed because the unchanged environmental condition compared to the other two times made it insignificant for the purposes of the study). Two techniques were used for the measurements: a) exposure for 30 min of 10-mm diameter Petri dishes containing culture media, both simple and selective for the various bacterial and mycotic species under investigation, arranged according to Fischer's I/l/1 pattern (27, 28) in pre-established points, in controlled and reproducible experimental conditions (doors and windows closed, aerators off, etc.); and b) the forced aspiration Surface Air System (29) with one-minute air-sampling for each microbiological index investigated, for the detennination also of the environmental microbial charge.
Periodic checks were made on staff, with oropharyngeal swab tests and faeces analyses.

After medication, balneotherapy tubs were washed with 5 litres of sterile physiological solution; equal parts of one litre of sample were then filtered through millipore membranes (0.45 pm) and placed on the surface of dishes containing the same culture media as mentioned above.

The nineteen patients altogether yielded 88 biopsies, 79 surface swabs and 51 sterile gauzes (for mycetes, research); 67 of the 88 biopsies were also used for histological examiliation.
Table I reports the results of the research for microbial flora in the various materials, in one or more samples examined after one week. This research showed that the species isolated with greatest frequency in the burn surfaces (biopsies and/or surface swabs) were Pseudomonas aeruginosa and Staphylococcus aureus, in respectively 78.9 and 47.4% of the patients; in six of these (31.6%) the two species were present simultaneously. Proteus was found in 26.3% of cases (P. morganii and P. mirabilis three times and P. mirabilis twice), always together with P. aeruginosa, while other enterobacteria (E. coli, Klebsiella, Epterobacter) and S. jaecalis were found only occasionally. C. albicans was isolated in one patient, both in the burn area and in the oropharynx.
The results of the biopsies and the surface swabs did not always correspond: in the latter, the bacterial species isolated were more numerous in 7 of the 19 cases examined. of types P1 I and P12 respectively 9 and 8 times; in three patients two serotypes were identified together.
It is interesting to note that both serotypes were present in 1989-90, and only P1 I (present in five out of the eight patients examined) in 1991-92. Other serotypes were occasionally present (Pl, P3 and P16).
As said, the biopsies of the 19 patients were also prepared for quantitative research of the microbic flora present as ' well as for qualitative research. Table II gives the results of microbiological monitoring

Three patients gave positive results for haemoculture (twice for P. aeruginosa and once for S. aureus); in another patient Pseudomonas was also isolated from the venous and urinary catheters.
The serological typing of the strains of P. aeruginosa performed with agglutination tests with specific antisera (provided by the Institut Pasteur) permitted identification performed at weekly intervals in three patients. These results are representative of the entire study and they relate to the microbial charge expressed per gm of tissue and to the bacterial species isolated. The first thing to note is that biopsies effected at different points - though very close together - in the same burn area sometimes yielded extremely variable microbial charge values (from < 10 colonieslgm to over 1 x 101 in Plate Count Agar). Even if the differences were not always extreme, the values were often dissimilar. In two of the three patients (Nos 12 and 17, and particularly in No 12) it is possible to note a progressive reduction in the number of colonies in the last tests, even if this finding did not always correspond to a qualitative improvement.
It is interesting to note that the species identified, Pseudomonas and S. aureus, were present at the beginning of hospitalization and persisted even after several weeks, despite the local and general treatment administered. Both species were nearly always found, either alone or in association, in all the areas examined and in the various test points.
The histological examination of the 67 biopsies from eight patients, stained with haematoxylin-eosin, made it possible to distinguish the various tissue levels at which it was not possible to observe bacterial elements.
In five cases, however, all fourth degree involving the deep hypodermis, it was possible to observe the presence of myeetes in the form of blastospores and hyphae; C. albicans was culturally isolated from the burn surface area in only one of these cases.

Environmental microbial monitoring was effected in the medication rooms and in the Intensive Care Unit. In each of the 42 tests (41 in the ICU) the air samples were taken at 8 a.m. and 12 a.m., and 2 p.m. after cleaning and disinfection procedures (no tests were made at 12 a.m. in the ICU) and examined for total microbial charge and for the presence of pathogenic and/or opportunistic microbial species.
Table III shows the values of microbial charge per in' of air, divided into the following four classes according to the degree of contamination: up to 5 and 15 UFC/ml (1st and 2nd class: operating rooms, wards with high-risk patients, etc.); and up to 75 and 100 UFC/m1 (3rd and 4th class: rooms at medium and low risk (22, 29).

Key: CFU = colony-forming units; * the number in parentheses indicates the number of tests
The two types of environment appear to present a different situation. In the medicating rooms the microbe levels at 8 and 12 a.m. were almost unchanged and remained within the first two classes in 60-65% of the samples. Conditions were better at 2 p.m., i.e. after cleaning procedures, and in particular there was a significant increase in the number of class I samples (0-5 UFC/m~ of air) from about 4-7% to over 28%. On the contrary, in the ICU, microbial charges - which at 8 a.m. registered a percentage of class I and 2 samples similar to that observed in the medication rooms, with a considerable increase in class 1 (21.9%) - did not present particular variations in the measurements effected at 2 p.m.
This different behaviour is confirmed by the results given in Table IV, which refer to the presence of one or more pathogenic and/or opportunistic microbial species in the air samples taken before and after cleaning of the rooms. As can be seen, the negative samples in the medication rooms increase from 7 to 23, while positive samples for two or more species reduced from 14 to 3; in contrast, the results in the ICU are almost identical in the two tests. S. aureus was the most resistant in both types of environment examined, and was still present in over half the samples taken after cleaning operations, while the persistence of P. aeruginosa and Acinetobacter was almost constant in the ICU and dropped considerably in the medicating rooms.

Key: * the number in parentheses indicates the number of tests

Table V shows the frequency of isolation of the various microbe strains from the nineteen patients compared with the frequency observed in the medication rooms and the ICU. S. aureus was found in 47.4% of the patients and in 60-70% of the air samples, while P. aeruginosa was detected in 78.9% of the patients and on average in about 14% of the air samples. The enterobacteria group (E. coli, Klebsiella, Enterobacter and Proteus) showed uniformity of presence, while Acinetobacter and filamentous forms of mycetes (Aspergillis, Penicillium, and Alternaria) were typically environmental.
It is important to note that in various tests P. aeruginosa of the same serotype and S. aureus were isolated simultaneously from the patient and from the air of the patient rooms.

Key: * the number in parentheses indicates the number of samples examined

Oropharyngeal swabs were taken periodically from the medical and nursing staff. S. aureus was isolated from six persons, two of whom also had type 6 P. aeruginosa; three others presented Klebsiella (twice) and E. coli.

Tests were made after daily medications, using dishes with different media for contact with the taps and for different points in the base of the tub. Type 12 P. aertiginosa was isolated in 11 of the 24 samples taken (45.8%), and S. aureus and Acetinobacter in respectively three and four cases. Repeated examination of the water, filtering 5 litres per millipore (0.45 pm) was always negative.

The results of microbiological investigations performed on a sample of 19 severely burned patients admitted to the Bari Polyclinic Burns Centre in the period 1989-92 indicated, as also found by other researchers (33, 38), that the aetiology of infection is due mainly to P. aeruginosa and S. aureus, which were found in respectively 78.9 and 47.4% of cases. These strains were present in patients from the first days of hospitalization. They were responsible for colonization of the superficial and deep tissues in the burn sites, and sometimes spread systemically, as found in three cases (results repeatedly positive to haemoculture). In addition, the persistence of P. aeruginosa in particular at the level of various burned sites, which was probably partly due to the ineffectiveness of the usual topical and antibiotic therapies (20), was often responsible for the failure of skin grafts to take, as reported elsewhere in the literature (7, 12, 37).
The fifteen carriers of P. aeruginosa yielded 21 strains, of which 17 (81 %) belonged to serotypes 11 (nine strains) and 12 (eight strains). The chronological distribution of these two serotypes was different: they were both present in the first part of the study, with a clear prevalence of P12, while only P1 1 persisted in the successive period. The high frequency of isolation of these two scrotypes from the nineteen patients examined, respectively 47.4 and 42.1%, is consistent with analogous observations in the literature (1, 8, 10, 17) of epidemic episodes or sporadic cases in hospitalized patients, some with severe bums, occuning at different times and caused by either of the two scrotypes.
Pitt et al. (25), after using various parameters (bacteriophagic typing, pyocyanin typing, electrophoresis of membrane external proteins) to examine strains of serotype 12 originating from various parts of Europe, suggested that they might have a common origin.
The patient biopsies were used, as said above, not only for qualitative examination but also for the determination of the microbial charge per gm of tissue. The importance of a quantitative assessment of the degree of infection of a burn wound has been widely recognised, especially as a clinical and prognostic index. Lindenburg et al. (18), as long ago as 1965, followed by Loebl (19) in 1974, found that a charge of 101 or more per gm of tissue was associated with severe infection and probable sepsis. In the cases reported by Volenee et al. (39) and Tahlan et al. (35), signs of sepsis, with worsening of prognosis and difficulty of skin grafts to take, appeared when the number of micro-organisms was 101- 101.
In the patients we examined, high microbial charges -even over 101 - were not generally associated with a state of sepsis; haemocultures were repeatedly positive in only three cases (two for P. aeruginosa and one for S. aureus). More than half the patients showed a reduction in microbial charge after some weeks of hospitalization. In the other patients the microbial charge remained high even for long periods, confirming the ineffectiveness of topical and antibiotic agents against the two bacterial species most cornmonly present, i.e. P. aeruginosa and S, aureus. Quantitative analysis was not however consistent throughout the various bioptic points in the same burn area, with microbe levels varying considerably even in adjacent sites. This was probably due to the presence of foci with particularly favourable conditions for the multiplication of microbes (e.g. presence of eschar). It is thus advisable to perform multiple biopsies in order to be able make an accurate and significant assessment of the degree and type of infection.
C. albicans had a very low isolation rate, being found in just one patient (5%) in a surface swab without any qualitative or quantitative bioptic confirmation. Prasad (30), in an ample number of cases, reported 11.6% Candida positive biopsies, with some fairly high charges. It should be noted however that in our study the examination of the histological samples, in this patient and four others with negative culture results, revealed the presence of mycetes in the deep dermis strata. It would therefore seem useful to submit full-thickness biopsies also to histological tests (13, 14, 21), which would represent a valid support for the culture tests, especially in cases requiring appropriate preparation of the bed of the lesions to be reconstructed.
P. aeruginosa and to a greater extent S. aureus were observed in air samples from the medication rooms and the ICU, often contemporaneously with their isolation from a patient, thus confirming the close relationship between environment and person, particularly in such areas of very high infective risk. The air contamination levels were different in the two types of environment considered. In the medication rooms, 35-40% of samples taken during routine nursing activities with the patients showed values exceeding the limit fixed for high~risk environments (i.e. 15 microbial units per M3 of air), with some improvement after cleaning and disinfection procedures which reduced the number of unfavourable samples and of pathogenic and/or opportunistic species. In the ICU, which clearly is intended for the long-term hospitalization of severely burned patients, there were no differences between samples taken before and after cleaning of the rooms, the levels being very similar to those of microbial contamination.
S. aureus was the species most regularly found both in the two types of environment and in patients (respectively in 60-70% and about 47% of the samples). P. aeruginosa was found in 79% of the patients but in only 14% of the air samples, thus confirming that this species in the hospital rooms is "human" rather than environmental. It is also interesting to note the repeated isolation of serotype 12 P. aeruginosa from the tap and the base of the balneotherapy tub, but never from the water, as also reported in other investigations (34). The balneotherapy installations therefore constitute a reserve of micro-organisms which derive from the patients and can be very easily transmitted to other patients.
Our findings, as also confirmed by various other investigations (33, 34), indicate the importance in a Burns Centre of systematic microbiological surveillance of the patients, staff and environment, considering that the aetiopathogenesis of infection in severely burned patients involves both host- and environment-related factors. Infection can be considered an inevitable stage in the burn disease and it is of basic importance to clarify the nature of the actiological agent that causes it, in order to avoid compromising the success of any reconstructive treatment or aggravation of the prognosis of the patients. Recent studies (16, 36) have stressed the advisability of postponing elective reconstruction treatment if the lesion bed is contaminated, particularly

RESUME. Les auteurs, après avoir examiné les difficultés de la thérapie antimicrobienne, analysent certains aspects récemment observés dans le traitement de la sepsis, c'est-à-dire l'utilité de classifier les patients brûlés selon des classes homogènes, les particularités des pathogènes, et les méthodes les plus efficaces pour les combattre. Ils proposent l'emploi de l'Index de Sévérité des Brûlures de Roi, qui permet de classifier les patients pour créer des groupes homogènes. Pour ce qui concerne la thérapie il semble que ni la thérapie antibiotique prévue ni la prophylaxie antibiotique ne soit utile, même si la prophylaxie à dose unique est indiquée en certaines conditions cliniques. Les auteurs conseillent l'emploi de vancomycine et de teicoplanine contre le Staphylococcus résistant à méticilline, et des aminoglycosides, conjointement avec la pénicilline, contre Pseudomonas aeruginosa.

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