STAPHYLOCOCCI IN THE HOSPITAL ENVIRONMENT

A. T. WELLS AND G. C. -* Department of Bacterwlogy, School of Medcine, Lee&

THa term " hospital staphylococcus " is used to define those strains of Staphylococcus mas which, with increasing frequency in recent years, have caused infections of surgical wounds, and pneumonia and gastro-enteritis in patients after their admission to hospital. These staphylococci are almost invariably resistant to penicillin, and often to streptomycin and the tetracyclines as well, and among them certain phage types of group I and group 111 predominate (Williams, 1959). Recently, other characteristics of hospital staphylococci have been reported. Moore (1960) showed that staphylococci of phage types commonly associated with epidemic surgical sepsis are more resistant to mercuric salts than nonepidemic strains. Turner and Willis (1962) found that the majority of staphylococci that are multiple-antibiotic- resistant and mercury-resistant form yellow colonies on glycerol monoacetate agar quite different from the orange- and creamaloured colonies of the more sensitive strains of Staph. aureus. They suggested that this yellow pigmentation is a marker characteristic likely to be of value in tracing the source of an outbreak of hospital infection when the causative organism is a multiple-resistant staphylococcus. They also suggested that " yellowness " may be a characteristic of staphylococci that are better adapted than others to cause cross- infection amongst patients in a hospital ward.

In this investigation we determined the mercury sensitivity and the pigmentation on glycerol monoacetate agar of 503 strains of Staph. auras freshly isolated from a general hospital, and examined the association of these reactions with the antibiotic sensitivity patterns, phage types and sources of the organisms.

MATERIALS AND METHODS

Struins ofstaphylococci. These were divided into four groups: (1) 120 strains isolated from one surgical ward during one month (13 Sept.-l3 Oct. 1961) when swabs were obtained from all post-operative surgical wounds, and from the noses of patients and staff, and samples were taken from the ward environment (air, bed covers); (2) 222 strains isolated consecutively during a period of 5 wk from specimens sent to the hospital laboratory from all in-patients, including exudate from septic lesions, nasal swabs and faeces; (3) 78 strains isolated con- secutively during a similar period from specimens sent from out-patients; these were chiefly swabs from superf~cial pyogenic lesions and urines; (4) 83 strains isolated from the noses of the staff of the bacteriology laboratory that receives

* Present address: Sefton General Hospital, Liverpool. 15. I. PA= MCL-VOL. 8s (1963) 395

396 A. T. WILLIS AND G. C. T U W E R

specimens from the hospital, but is situated in a separate building remote from the wards. Nasal swabs were taken from 41 individuals usually on 5 occasions, at weekly or fortnightly intervals, but owing to some absences only 191 swabs were taken. This was a useful group for comparison because it consisted of healthy individuals who did not work inside the hospital.

Surgical ward. In the investigation of the surgical ward the methods used were, in general, those of Turner and Willis, who described a similar investigation on the same ward which ended 2 mth previously. This ward contains 24 beds (fig. 1) and deals with the usual range of general surgical procedures with special emphasis on surgery of the colon and rectum. The day staff consists of a sister and 7 nurses; at night there are 2 or 3 nurses. Bedding consists of linen, cotton cellular blankets and cotton bedcovers, all of which are changed and boiled weekly or when patients are discharged.

Isolation of staphylococci. In the surgical ward, during 4 weeks, swabs were taken from operation wounds at the first dressing, and subsequently if there was evidence of infection. Nasal swabs were taken from patients and staff on the first day and 2, 3 and 4 wk later. At the same time, sweep-plate cultures were

M FIG. 1.-Plan of surgical ward. Black rectangles indicate beds; black circles indicate

settle plates.

made from each patient’s bed-cover, and settle plates, to recover organisms from the air, were exposed From 10 a.m. to 4 p.m. in 26 sites in the ward (fig. 1).

Brain-heart infusion agar containing 1 per cent. of glycerol monoacetate (Willis and Turner, 1962) was used for inoculation of swabs, and for settle plates; this medium with the addition of 5 per cent. sodium chloride was used for sweep- plate cultures. After 48 hours’ incubation at 37°C. cultures were examined for colonies of staphylococci. The colour of coagulase-positive staphylococci was recorded, and colonies were subcultured on to glycerol monoacetate agar for purity and for confirmation of pigment variety.

Specimens from other hospital patients (groups 2 and 3) were initially plated on meat infusion agar, blood agar or salt agar, depending on the nature of the specimen, and were subcultured to ensure purity. Nasal swabs from members of the staff of the bacteriology laboratory were inoculated directly on to glycerol monoacetate agar.

Test for pigmentation. After 48 hours’ incubation on monoacetate agar it was possible to determine whether pigmentation was yellow or ‘‘ orange ”, the latter category including deep orange, pale orange and creamcoloured pigmentation. This colour differentiation was observed in primary culture and all subsequent cultures on glycerol monoacetate agar, but was never seen on the other media used.

Phage typing was camed out by Dr G. B. Ludlam. Antibiotic sensitivity tests were performed on meat infusion agar using Multo-

disks (Oxoid) at the following concentrations: penicillin, 1-5 units; streptomycin, tetracycline, chloramphenicol and erythromycin, 10 pg.; novobiocin, 5 pg. All

STAPH YLOCOCCZ IN HOSPITA 397

strains were tested for sensitivity to methicillin (Celbenin, Beecham Research Laboratories) with 10 pg. discs.

Mercury semitivity test. Moore recommended spot-inoculation of overnight broth cultures on to freshly prepared peptone agar containing 1 in 27,500 mercuric chloride. Staphylococci that grew in 12-18 hr were regarded as mercury-resistant. We have found this method unsatisfactory because, as Moore showed, mercuric chloride is rapidly inactivated in the medium so that plates cannot be stored for more than 3 days. We have developed a disc method which has the advantage that ordinary nutrient agar plates can be used.

Preparution of mercuric chloride discs. Discs 6 mm. in diameter were punched from sheets of Whatman No. 1 lilter paper and were dry-sterilised in freeze-drying tubes in batches of 500. A 1 in lo00 solution of mercuric chloride was prepared by dissolving a tablet of mercuric chloride, BPC, in 600 ml. distilled water. The solution was added in 5 ml. volumes to batches of 500 discs. The whole volume of fluid was absorbed (see Gould and Bowie, 1952) so that each disc contained about 10 pg. mercuric chloride. The discs were then freeze-dried by the single- stage method described by Flewett ef ul. (1955), transferred to sterile screwcapped bottles and stored at room temperature. Similar control discs, impregnated with 0.9 per cent. sodium chloride, were prepared.

RE!suLTs Disc test for mercury sensitivity

In a preliminary experiment, the mercuric chloride discs were used to test 143 strains of Staphylococcus aureus, 90 of which were known to be mercury-resistant, and 53 mercury-sensitive, by Moore’s method. Each st ra in was inoculated on meat infusion agar and the mercuric chloride and control discs were placed on the surface; at the same time, each strain was re-tested by Moore’s technique. The results were read after overnight incubation at 37” C. Strains that failed to grow on Moore’s medium (i.e., that were mercury-sensitive) showed zones of inhibition 10 mm. or more in diameter with 10 pg. mercuric chloride discs, whilst strains that were mercury-resistant on Moore’s medium showed either no zones of inhibition or zones less than 10 mm. in diameter. The sodium chloride control discs were inactive. Mercuric chloride discs retained their activity when stored at room temperature for up to 3 mth. The most satisfactory discrimination between mercury- sensitive and mercury-resistant strains was achieved with discs con- taining 10 pg. mercuric chloride, and these were used for the subsequent tests.

Relationship of mercury sensitivity to antibiotic sensitivity Each of the 503 strains of Staph. aureus isolated in this survey

was tested on a single plate of nutrient agar by the disc technique for sensitivity to mercuric chloride and antibiotics. The results are summarised in table I. All the strains were sensitive to chloramphenicol, erythromycin, novobiocin and methicillin.

Only 4 of the 144 antibiotic-sensitive strains were resistant to mercuric chloride; and of 117 strains resistant to penicillin but sensitive to tetracycline, only 6 were resistant to mercury. On the other hand,

398 A. T. W U I S AND G. C. TURNER

of the 242 multiple-antibiotic-resistant strains, 234 were mercury- resistant. Of the 8 tetracycline-resistant strains that were sensitive to mercury, the 5 in the laboratory series were all from the same individual.

TABLE I Mercury and antibiotic sensitivity in strains of Staphylococcus aureus

isolated fiom diferent sources

resistant sensitive to !o. antibiotics* pemcillin

only

-- Source of strains multiple

antibiotic- resistantt I I

Ward survey speci-

In-patient specimens Out-patient speci-

Laboratory staff nasal swabs

mens

mens

sensitive to antibiotics+

Allsources .

resistant multiple-

only resistant? to penicillin antibiotic-

No. of mercury-sensitive strains

17

31 34

58

15 1

47 2 29 0

m 5

?I--

I Mercury-sensitive Mercury-resistant

strains strains Reaction to tetracycline

No. (percent.) No. (percent.)

Total no. of strains

l 8 140 I 111

Sensitive . . . Resistant . . .

No. of mercury-resistant strains

251 (96.2) 10 (3.8) 261

8 (3.3) 234 (96.7) 242

* Sensitive to penicillin, streptomycin, tetracycline, chloramphenicol, erythromycin,

t Resistant to penicillin, streptomycin and tetracycline. novobiocin and methicillin.

Thus, with few exceptions, mercury resistance and tetracycline resist- ance ran parallel (table 11). It is also interesting to note (table I) that 221 of the 234 strains that were resistant to both mercury and tetracycline were isolated in the ward survey or from in-patient

specimens. These results support our earlier finding (Turner and Willis) that the chief value of the mercury test is that it identifies the multiple-antibiotic-resistant staphylococci that are normally recognised by routine antibiotic sensitivity tests.

STAPHYLOCOCCI IN HOSPITAL 399

Pigment production and antibiotic sensitivity The relationship between pigment production on glycerol mono-

acetate agar and antibiotic sensitivity in these staphylococci is shown in table III. Altogether 341 orange strains and 162 yellow strains were isolated. None of the yellow strains was sensitive to all the antibiotics and only 4 were sensitive to tetracycline (table III). On

TABLE 111 ReIatwnship of pigmentation to antibiotic sensitivity in Staph. aureus

strains from different sources

In-patient specimens Out-patient speci-

mens Laboratory staff

nasal swabs

No. of orangepigmented strains I

31 34

58

source of strains sensitive to antibiotics'

Ward survey: Staffnoses . Patients noses . Septic lesions . Ward environment All sources in ward

3 8 2 8

21 I

Allsources . . I 144

resistant to

penicillin O d Y

5 4 2 4

1s

49 29

20

113

multiple antibiotic- resistantt

No. of yellow-pigmented strains

I I

O F 4 158 (459

0

0

Total no. of Strains

13 27 11 69

120

222 78

83

503

* Sensitive to all antibiotics tested (see footnote, table I). Resistant to penicillin, streptomycin and tetracycline.

$ No. of strains of phage type 53+ given in brackets. 8 No. of strains of phage type 77+ given in brackets.

the other hand, 144 orange strains were antibiotic-sensitive and 113 were resistant to penicillin but sensitive to tetracycline. Of the 242 tetracycline-resistant strains, 158 were yellow and all but 12 of these were isolated from the ward-survey or in-patient specimens. Thus, with few exceptions, " yellow " staphylococci formed a major sub- division of the multiple-antibiotic-resistant group.

The ward survey When the investigation began on 13th Sept. 1961, there were

24 patients in the ward, and 44 were admitted subsequently during the period under review. Of the 68 patients, 55 underwent surgical operations, and Staph. aureus was isolated from new post-operative sepsis in 6 of these; in addition, one patient developed staphylococcal sepsis in a wound previously infected with coliform organisms.

400 A . T. WILLIS AND G. C. TURNER

A total of 120 strains of Staph. a u r a was isolated from all sources in the ward. All of 27 yellow strains and 57 of 93 orange strains were multiple-antibiotic-resistant. Each of these two multiple-resistant groups was dominated by one phage type; 22 of the 27 yellow strains were of type 77+ and 53 of the 56 orange strains were of type 53+ (table 111). The other resistant strains and the sensitive group included many different phage types. Table I11 also shows the relationship between pigmentation and sensitivity pattern and the sources from which the organisms were isolated. The inoculation of nasal swabs directly on to glycerol monoacetate agar enabled us to detect, in the case of 4 individuals, simultaneous nasal carriage of two different strains of Staph. aureus. In each case, yellow and orange varieties were recognised and found to be of different phage types. We have, however, also encountered distinct and stable yellow and orange strains of the same phage type.

The proportion of yellow multiple-resistant strains was much higher among staphylococci isolated from septic lesions than among those from either patients’ noses or the ward environment (i.e., air and bed-covers). To assess the significance of this finding, it is necessary to consider the incidence of sepsis in the ward and the frequency of isolation of the different varieties of staphylococci from the septic lesions and other sources (fig. 2).

On 13 Sept., 47 per cent. of the samples from the ward environ- ment yielded Staph. aureus and all these were multiple-resistant orange strains of type 53f. We were not able to discover the source of this “ broadcast ” since the organism was not recovered at this time from the noses of staff or patients or from any septic lesions. The only case of open sepsis then in the ward was a patient admitted on 2 Sept. with a carbuncle of the face; at this time, a swab from the lesion yielded a Staph. aureus that was not phage-typed but was sensitive to all antibiotics tested except penicillin. The infection persisted and when the lesion was swabbed again on 21 Sept., a multiple-resistant yellow staphylococcus of type 77f was recovered. Samples from the ward environment obtained on 27 Sept. and 4, 10 and 13 Oct. showed a steady decline in the isolation rate of staphy- lococci. This decline was attributable to the diminishing numbers of organism of type 53+. During the last week of the investigation, however, the isolation rate increased owing to the appearance of strains of type 77+ which had not previously been encountered in the environment.

Despite the numerous isolations of orange type 53+ staphylococci from the environment, only one case of infection by this organism occurred. This was in a patient aged 77, admitted on 21 Sept. with obstructive jaundice and found subsequently to have diarrhoea; on 27 Sept., her faeces yielded large numbers of type 53+ staphylo- cocci. This patient, three other patients and one nurse became nasal carriers of the same organism.

STAPH yu)COCCI IN HOSPITAL 401

Although several cases of wound infection occurred in which the organism responsible was the yellow type 77+ staphylococcus, this organism was not recovered from the ward environment before 10 Oct. One patient who had undergone prodocolectomy developed staphylococcal pneumonia at the same time as the wound infection, and staphylococci of type 77f were isolated from both the perineal

Sqhrlaocc i . . . -. . - - * . . - isolated from scptk fcsions

..... .... ‘-*

and bed samples yielding

Stoph. oureus

20

lo t 13 k p t . 27 Scpr. 4 Oct. 10 13 Oct.

Date of sampling FIG. 2.-Distribution of Staphylococcus w e u s in the ward environment in relation to

staphylococcal sepsis,

incision and the sputum. It seems likely that this patient was responsible for the dissemination of type 77+ organisms into the environment.

Only two patients developed sepsis due to mercury-sensitive staphylococci. In one, in whom an abscess developed at the site of a femoral ligation of varicose veins, the organism responsible for the infection was also recovered from the patient’s nose but only once from the environment. An organism of the same type, however, was isolated from a small septic lesion on the forearm of a nurse. In the second patient an organism of another phage type was isolated from an anal fistula but was not encountered elsewhere.

In three of the infections staphylococci of the same phage type were recovered from patients’ noses ufrer the infection developed, although previously nasal swabs had yielded either no Staph. aweus or staphylococci of different phage types.

In this short series, therefore, yellow staphylococci predominated J. PATB. MCK-VOL. 8S (1963) 2c

402 A. T. WILLIS AND G. C. TURNER

among those causing infection although they were not recovered from the ward environment until after several cases of sepsis had occurred. This was in accord with our previous finding on the same ward (Turner and Willis), but did not necessarily indicate that yellow strains pre- dominate generally in cross-infection, since all the yellow strains that caused sepsis in this series were of one phage type.

6 8

0 3 2

In-patient specimens Table IV summarises the distribution of staphylococcal varieties

isolated from in-patient specimens. The incidence of tetracycline- resistant staphylococci was 140 (63 per cent.), of which 119 (85 per

TABLE IV Tetracycline sensitivity and pigmentation in strains of Staph. aureus

isolated from different sources in in-patients

48 29

9 4

21

Source of strains

. . ..

Wounds Sputum, mouih and

Faeces. . . Nose . . . Urine . . . Skin . . . Others . . .

throat

Allsources .

No. of tetracycline- resistant strains

I

q 7 1

21 1 119

No. of tetracycline- sensitive strains

Orange Yellow I 0

3 1 0 1: 1 0

1 I 1

@ ) I 2

Total no. of strains

73 57

9 23 26 16 18

222

cent.) were yellow; among the 82 tetracycline-sensitive strains, how- ever, only 2 (2.3 per cent) were yellow. Among the tetracycline- resistant strains, yellow varieties did not show a predilection for any particular type of infection. Yellow staphylococci were fairly evenly distributed among infected wounds and urines and those miscellaneous specimens (" Others ", table IV) which included pleural fluids, blood cultures and eye swabs. From specimens of faeces in this series, yellow staphylococci were the only ones isolated, but the number of specimens examined was too small for this to be a significant finding. Most of the strains from skin infections were tetracycline-sensitive.

Out-patient specimens Table I11 shows that of 78 strains of Staph. aureus isolated from

infections treated in the casualty and out-patient departments, 65 were tetracycline-sensitive, 63 of these being orange. Of the 13

STAPHYLOCOCCI IN HOSPITAL 403

tetracycline-resistant staphylococci, 12 were yellow and 11 of these were from recent in-patients, and were probably of hospital origin.

In addition to the staphylococci isolated in the ward survey, the multiple-resistant staphylococci isolated from in-patient and out- patient specimens were all phage-typed. They were found to include many different phage types and most of these belonged to group 111; less than 20 per cent. were of the yellow type 77 + and none was of the orange type 53 + which predominated in the ward survey.

Nasal swabs from laboratory staff Forty-three per cent. of these swabs yielded Staph. aweus. Most

of the strains isolated were penicillin-sensitive and this was clearly a typical non-hospital group. No yellow staphylococci were encoun- tered, and the 5 multiple-resistant orange strains were those, all isolated from one individual, that were exceptional in being sensitive to mercury.

DISCUSSION The antibiotic sensitivity tests of the various groups of staphylo-

cocci showed, as we expected, that most strains from the nasal swabs of laboratory staff and many of those from out-patient specimens were sensitive to all antibiotics. Nearly all the remaining strains in these two groups were resistant to penicillin only. On the other hand, staphylococci from in-patients and those isolated in the ward survey (from patients and the ward environment) included a high proportion of organisms resistant to penicillin, streptomycin and tetracycline. Relatively few were sensitive to penicillin.

Nearly all strains sensitive to all the antibiotics, or resistant to penicillin only, were sensitive to mercuric chloride. By contrast, most tetracycline-resistant staphylococci, including many different phage types, were also mercury-resistant. Mercury resistance is thus a characteristic closely associated with multiple antibiotic resistance. It is not surprising that Green (1962) was unable to correlate mercury resistance with penicillin resistance because it is the strains that are resistant to both penicillin and tetracycline that are also mercury- resistant, whilst strains resistant only to penicillin are mercury-sensitive. If, therefore, in the investigation of an outbreak of epidemic sepsis, it is not possible to perform antibiotic sensitivity tests on all the staphylococci isolated, the mercury test may be of value in picking out the multiple-antibiotic-resistant strains, which are the ones most commonly involved in hospital infection. Since, however, the cor- relation between mercury and tetracycline sensitivity is not absolute (three of our hospital isolates were mercury-sensitive but tetracycline- resistant) the mercury test cannot be accepted as an alternative to antibiotic sensitivity testing.

Strains that formed yellow pigment on glycerol monoacetate agar were nearly all multiple-antibiotic-resistant and they accounted for

404 A. T. WILLIS AND G. C. TUMER

87 per cent. of the multiple-resistant staphylococci isolated. Nearly all these yellow strains were isolated from patients in the hospital or from out-patients with recent hospital experience. A few yellow strains were recovered from the noses of ward staff, but none was isolated from the laboratory staff despite the fact that the latter were handling specimens and cultures that sometimes contained “ yellow ” staphylococci.

These yellow staphylococci are thus a subdivision of the multiple- antibiotic-resistant group and their distribution strongly suggests that they have become prominent in hospital cross-infection since the introduction of antibiotics, especially the tetracyclines. As Williams et al. (1960) pointed out, resistance to tetracyclines does not often occur in a patient de novo, but is usually due to cross-infection with a new strain. Such cross-infection usually occurs from the noses or septic lesions of other patients, situations in which we have found that the great majority of multiple-antibiotic-resistant staphylococci are yellow. These considerations may explain why the yellow staphy- lococci studied in our ward survey, and those studied by Turner and Willis, played a part in hospital cross-infection out of all proportion to the numbers of multiple-resistant staphylococci recovered from the ward environment. It is interesting to note in this connexion that most of our yellow staphylococci belonged to phage-group 111, strains of which acquire resistance to antibiotics other than penicillin more easily than those of other groups.

There is no doubt that pigmentation on glycerol monoacetate agar is a valuable marker characteristic. In the investigation of an outbreak of hospital infection due to a yellow staphylococcus the search for the offending organism can be confined to organisms forming yellow colonies on glycerol monoacetate. The “ orange ” strains include organisms, the colonies of which are deep orange, pale orange or cream, and these appearances are often variable in sub- culture. In a staphylococcal “ broadcast ”, however, we have found that, among the “ orange ” strains, those that are identical can be recognised by the similarity of their pigmentation on primary isolation. Thus, Turner and Willis recorded a staphylococcal broadcast that was due to a deep orange tetracycline-sensitive strain. The various orange varieties are quite distinct from those forming yellow pigment.

On a number of occasions we have observed orange and yellow varieties of the same phage type (see also Willis and Turner). These pigmentation varieties are quite stable in artificial culture and are clearly different organisms. In looking for the source of an outbreak of staphylococcal sepsis, therefore, reliance cannot be placed on phage-typing alone ; the pigmentationvariety should also be determined.

SUMMARY An examination of 503 strains of StaphyZococcus uureus isolated in

a general hospital showed that most of those that were resistant to

STAPHYLOCOCCI IN HOSPITAL 405

penicillin, streptomycin and tetracycline (i.e. " multiple-resistant ") were also resistant to mercuric chloride. These strains were the predominant cause of staphylococcal sepsis in hospital in-patients, were uncommon causes of staphylococcal infections in out-patients, and were not isolated from the noses of normal healthy individuals. Most multiple-resistant strains isolated from in-patient infections formed yellow-pigmented colonies on glycerol monoacetate agar, a characteristic rarely seen in sensitive strains. On the same medium the colonies of other multiple-resistant strains were " orange ".

These colour varieties of Staph. aureus on monoacetate agar provide useful marker characteristics for the recognition of epidemic strains causing hospital outbreaks of surgical sepsis. Tests for pigment production and antibiotic sensitivity can be used to follow the develop- ment of staphylococcal " broadcasts " before this is retrospectively studied by phage typing.

We are indebted to Professor C. L. Oakley for his interest and encouragement in this work, to Professor J. C. Goligher and Sister D. R. Lynas for their active cooperation, and to Dr G. B. Ludlam for the phage typing of our staphylococci.

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J. PAM. BACT.-VOL. 85 (1963) 2 c 2