Surgery Today Jpn J Surg (1993) 23:947-953 ~ SURC~RYTO~v © Springer-Verlag 1993

Original Articles

Methicillin-Resistant Staphylococcus Aureus Infections Following Esophageal Surgery in Patients with Impaired Defense Mechanisms

TAKAO SAITO, TADAHIKOKINOSHITA, YUJI SHIGEMITSU,TAKESHI KATSUTA,KATSUHIRO SH1MODA, and MIcHIO KOBAYASHI The First Department of Surgery, Oita Medical University, Hasama-machi Idaigaoka, Oita, 879-55Japan

Abstract: This study was conducted to determine whether or in immunocompromised hosts, little is known of its not compromised host defense mechanisms prior to surgery possible correlations. are related to postoperative infections with methicillin-resistant Patients undergoing surgery for esophageal cancer Staphylococcus aureus (MRSA). Neutrophil cytocidal activities, are at an especially high risk of developing postoperative serum complement and immunoglobulin levels, the in vivo MRSA-related pneumonia. This risk of infection is antibody-producing capacity against pneumococcal poly- related to an increased rate of colonization of MRSA saccharide (PPS), and cell-mediated immunity (CMI) were in the respiratory tract, based on aggressive mea- evaluated in 22 patients who underwent esophagectomy for esophageal cancer between 1989 and 1990. Postoperatively, sures required in surgical intensive care units (ICUs) 8 nine patients developed MRSA infections. Anti-PPS IgG was including mechanical ventilation, 9 and is also probably found to be significantly lower in patients with MRSA infections attributable to impaired host defense mechanisms than in those without (P < 0.01). All the patients with MRSA against the pathogen, caused by severe surgical stress, infections showed a titer <600 EU, while all but one of the non- malnutrition, old age, and malignancy. 1°'11 infected patients showed a titer >600 EU. Impairment in other In order to determine whether a possible association components of the defense mechanisms, apart from a partial exists between the preoperative impairment of host deficiency of CMI, did not differ between the groups. Thus, a defense mechanisms and the development of post- preoperative evaluation of the antibody-producing capacity operative MRSA infections, and to identify the immu- may serve to predict the development of MRSA-related nological risk factors predisposing to MRSA infection, infections following major surgery such as esophagectomy. the host defense mechanisms, including the specific Key Words: MRSA, humoral immunity, polysaccharides, antibody-producing capacity to bacterial polysac- esophageal cancer, pneumonia charides were evaluated in patients with esophageal cancer.

Patients and Methods Introduction This survey was conducted between January, 1989 and Since the first documentation of methicillin-resistant December, 1990, when the outbreak of MRSA was Staphylococcus aureus (MRSA) strains in 1961,1 out- most prevalent in the surgical ward and surgical ICU of breaks of MRSA infection have been reported from Oita Medical University Hospital. Twenty-two patients Europe, the USA, Australia, and other countries. 2-4 with thoracic esophageal cancer underwent trans- In Japan, it has become prevalent since 1982. The thoracic subtotal esophagectomy during this 2-year characteristics of the epidemic strains, s epidemiology, period. There were 18 men and 4 women, aged between including the high endemicity in hospitals and carrier 44 and 82 years with a mean age of 65 + 8 years. states, 6 and control measures against the spread of the Disease stage was determined according to the TNM responsible organisms 7 have been well documented. classification of malignant tumors. 12 Informed consent Although MRSA infection is considered to occur for the study was obtained from each patient. Polymorphonuclear neutrophils (PMN) were purified Reprint requests to: T. Saito by the method of Boeyum. 13 Intracellular killing was (Received for publication on July 23, 1992; accepted on Jan. assayed by a modified version of the method of Leijh et 8, 1993) al. 14 and Shigemitsu et al. is using Staphylococcus 948 T. Saito et al. : MRSA Infection and Defense Mechanisms aureus (209p). The intracellular killing index (KI) was Nutritional status was assessed by measuring: body expressed as the percentage decrease in the initial weight, triceps skin fold thickness, mid upper-arm number of viable intracellular bacteria at 60 min. 15 The muscle circumference, creatinine-height index, serum superoxide anion producing capacity (SOP) of PMNs albumin, transferrin, prealbumin, and retinol-binding was assayed according to the method of Nakagawara protein, a5 A measurement was considered abnormal and Minakami.16 Phorbol myristrate acetate was added when it was: <90% of the standard for BW and AMC; as a stimulant and the rate of reduction in ferricyto- <70% for TSF; <60% for CHI; <3.5 g/dl for albumin; chrome C was measured and expressed as nmol/ <226mg/dl for transferrin; <21.0mg/dl for preal- 105 cells/rain. 15 Intracellular myeloperoxide activity bumin; and <2.5 mg/dl for retinol-binding protein. 25 (MPO) was measured by a modified method using O- Sputa was cultured on admission, then every week dianisidine, 15'17 and MPO activity was expressed as after surgery for at least 4 weeks. Tracheal aspirate 85.4 _+ 6.0%, 0.82 +_ 0.45 nmol/105cells/min, and 1.25 was cultured postoperatively twice a week in patients + 0.87 u/105 cells/min, respectively, is on a respirator by aseptic suctioning of the tracheobron- Serum IgG, IgA, IgM, C3, and C4 were quantitated chial tree. Wound secretions, blood, urine, and drain- by a rate nephelometric immunoassay. 18 The total age fluid were cultured as required. Organisms were hemolytic complement (CHs0) was examined by the identified according to routine microbiological methods method of Mayer. 19 Each parameter was considered in the central microbiology laboratory of our univer- abnormal when it was: <890 or >l,744mg/dl for IgG; sity hospital. Staphylococcus aureus was identified <82 or >363mg/dl for IgA; <52 or >298mg/dl for using colonial morphologic techniques, positive cata- IgM; <71 or >148mg/dl for C3; <9.5 or >32.8mg/dl lase, the coagulase test, and the Staphyslide test (Bio for C4; and <30 or >45 U/ml for CHs0 .2° Merieux SA, France). Susceptibility tests were per- The patients and healthy controls all received a formed according to the microdilution broth method single subcutaneous injection of 0.5 ml of the 23-valent using the MIC 2000 Frozen plate 'Eiken' (Eiken pneumococcal polysaccharide vaccine (PPS, Pneu- Chemical, Tokyo, Japan) and the MIC-2000 disposable movax, Merck, Sharp and Dohme, Quebec, Canada). inoculator (Dynatech Laboratories Va.). A minimal Peripheral blood samples were taken on days 0, 7, 14, inhibitory concentration (MIC) to oxacillin of >4 pg/ml and 21 after immunization, and the serum was stored at was defined as methicillin resistant. The trachea -30°C. Serum IgG, IgA, and IgM reactive with the was considered to be colonized with MRSA when PPS were measured using a modified enzyme-linked pathogens were cultured in the absence of any signs immunosorbent assay (ELISA). 21 The capacity of anti- of inflammation. PPS-specific antibody production was expressed as the Pre- and postoperative nutritional support was pro- peak titer between days 0 and 21 after immunization. vided either enterally or parenterally. All patients were The geometric mean of the peak levels of IgG, IgA, cared for in the surgical ICU during the initial post- and IgM in nine age-matched healthy controls, with a operative week while receiving at least 3 days' pro- mean age of 62 + 10 years, was 2.43 +~ 0.89, 1.78 +~ phylactic mechanical ventilation via an endotracheal 0.67 and 1.19 +x 0.51, respectively. tube connected to a mechanical volume ventilator. A Lymphocytes were collected using a Ficoll/Conray cutaneogastric tube, through a gastrostomy was used (Pharmacia Fine Chemical, Uppsala, Sweden) gradient. rather than a nasogastric tube, which was never used. T lymphocytes were counted by a sheep red cell rosette Antibiotics consisting of the 2nd generation cephems assay, 11'22 and B lymphocytes were identified by the and aminoglycosides were prophylactically prescribed. presence of surface IgG markers with the application of The postoperative course of each patient was carefully fluorescein-labeled antihuman globulin. 11'a3 Phyto- monitored.25 Pneumonia was diagnosed if a chest X-ray hemaglutinin (PHA)-stimulated blastogenic transfor- film showed new and persistent infiltration and at least mation was assayed by a modified version of the three of the following: (1) Purulent sputum, (2) an method of Oppenheim and Rosenthat. 24 Delayed cut- important respiratory or nosocomial pathogen isolated aneous hypersensitivity tests using purified protein from culture of the tracheal aspirate, (3) peripheral derivative (PPD) were performed by injection into the leukocytosis of >10,000 cells/ram 3, and (4) a fever volar surface of the forearm, and the erythema at >38°C. the injected site was read at 48h. ~a Each parameter For statistical evaluation, the Student's t-test, chi- was considered abnormal when it was: <1,363 or square test, and Fisher's exact test were used, with the >2,880 cells/mm ~ for the total lymphocyte count; level of significance being P < 0.05. <1,104 or >2,333 cells/mm 3 for the T-cell count; <85 or >346 cells/ram 3 for the B-cell count; <37,700 or >62,400 cpm for PHA transformation; and <10 mm for the PPD skin test. 11 T. Saito et al.: MRSA Infection and Defense Mechanisms 949

Results Table 1. Demographic features and risk factors. No features or risk factors reached a level of significance On admission, MRSA was not isolated from the Group I Group II Group III sputum of any of the 22 patients with esophageal (n=4) (n=6) (n=7) cancer. However, postoperatively, it was isolated from Age (years) 61 + 5 62 _+ 10 66 -+ 6 the tracheal aspirate or wounds of 17 patients, 9 of Sex (M:F) 4:0 5:1 5:2 whom developed an MRSA infection. According to TNM stage 0 0 0 0 the presence or absence of colonization and infection I 1 0 0 with MRSA, these patients were separated into the II 0 1 3 following three groups: Group I, being patients without III 2 4 4 IV 1 1 0 MRSA colonization (n = 5); group II, being those with Surgery MRSA colonization but no infection (n = 8); and group Duration (h) 10.4 + 1.9 9.0 + 2.8 8.6 + 2.2 III, being those with an MRSA infection (n = 9). Blood loss (ml) 1,462 + 606 1,046 + 694 1,036 + 445 MRSA was isolated in the tracheal aspirate of all the Antibiotics useda patients in groups II and III and also in the wound Cefmetazole 3 2 6 Flomoxef 2 4 3 secretions of two patients in group III, from the sodium subcutaneous phlegmon of the chest, and the anas- Aztreonam 0 0 1 tomotic site, respectively. The type of clinical MRSA Imipenum/ 0 0 2 infection in group III was pneumonia in eight patients Cilastatin sodium and a wound infection of the chest wall in one. MRSA Amikacin 4 6 7 was first isolated 1-7 days postoperatively in 9 patients, Tobramycin 0 0 1 8-14 days postoperatively in 13, and >15 days post- operatively in 4, while MRSA infection was first aThe prophylacticuse of antibiotics between surgeryand the time of first isolation of MRSA diagnosed 1-7 days postoperatively in 3 patients, 8-14 days postoperatively in 4, and 15-21 days post- operatively in 2. Clinical infection without MRSA did not occur in any The cytocidal activity of PMN before surgery was of the 22 patients, although one patient in group II had measured in 20 of the patients with esophageal cancer acute cholecystitis and three patients, being two in (Fig. i). The mean KI was significantly lower in the group III and one in group I, had anastomotic leakage. esophageal cancer patients than in the age-matched Mixed infections or colonizations of MRSA with other controls. However, there were no significant differences microorganisms were found in most patients. Seven in the mean K1 between the patients with MRSA of the nine patients in group III had more than infections (group III) and those without (groups I and two different species of bacteria cultured. Six patients II), or among groups I, II and III. Moreover, there developed pneumonia with Gram-negative bacteria, were no significant differences in SOP and MPO being Enterobacteriaceae and Pseudomonadaceae in between the esophageal cancer patients and the con- two, Enterobacteriaceae in two, and Pseudomonadaceae trols, or among the three groups. in two, and the remaining patient developed sub- Regarding the serum levels of complement com- cutaneous phlegmon with Gram-negative bacteria, being ponents (Fig. 2) and polyclonal immunoglobulins (Fig. Enterobacter cloacae and Gram-positive cocci, being 3), some patients showed higher values than the stan- Enterococcus faecalis. The tracheal aspirate from six of dard for C4, CH50 and IgA and lower values for IgM, the eight patients in group II was colonized with MRSA although there were no significant differences among and other bacteria, being Pseudomonadaceae in four the three groups. patients and Haemophilis influenza in two. The capability of anti-PPS antibody production was Demographic data and details of the recognized risk measured in 17 patients, while the remaining 5 refused factors for postoperative MRSA infections were com- to be immunized with PPS vaccine. The geometric pared among the three groups. There were no signi- mean of anti-PPS IgG, IgA, and IgM as a whole was ficant differences in sex, age, TMN tumor stage (Table 2.77 +x 0.63, 2.02 +x 0.60, and 1.33 +x 0.52, respectively, 1), underlying diseases, impairment of the vital organs and there were no significant differences between including pulmonary function, and nutritional status the patients and age-matched controls. The geometric (data not shown). Surgical stress factors, including the mean of anti-PPS IgG was significantly lower in patients duration of surgery and blood loss (Table 1), were with MRSA infections than in those without, being 2.29 similar. The perioperative management, being nutri- +x 0.33 in group Ill versus 3.10 +x 0.57 in groups I and II tional support, mechanical ventilation (data not shown), (P < 0.01). A similar tendency was observed for anti- and the prophylactic use of antibiotics (Table 1), were PPS IgA, being 1.74 ._.x 0.56 in group III versus 2.21 +x not significantly different. 0.54 in groups I and II (P = 0.13), although the 950 T. Saito et at.: MRSA Infection and Defense Mechanisms

~-~ 2 4

E E o 100 ol ~ 3 Fig. 1. Cytocidal activities of poly- 0 0 O O ~.I ...... 8.~ ..... x~... morphonuclear neutrophils (PMN) 0 / o; °ST in the patients without methicillin- o resistant Staphylococcus aureus "- 1 o 2 0 0 (MRSA) colonization (group I), those o 0 50 with MRSA colonization (group II), E o* 81 0 0 and those with MRSA infection (group O~ 0 O - III). Individual values and the mean _+ ~i O o 0 1 o~} O O SD of each group were compared for ...... O ...... ~o.L ol o superoxide anion producing capacity O 0 ...... 0 ...... (SOP), myeloperoxidase activity (MPO), and the intracellular killing I 11 llI I II llI I 1I Ig index (K/)

O

O O "" o ,-'-. 30 I! oo iTo+ "° ..... ~ .... °1~ .... ~ ~00 8 ~ ~ ol o°o° "° O'~ o o ~ ,oI,oI °i 0 o o~l o ~ E o E 20 O ~ ~ m Fig. 2. Serum complement levels in 212 ~ 50 ~ ~ 2~ the patients without MRSA coloniza- tion (group I), those with MRSA 10 ; ...... colonization (group II), and those with MRSA infection (group III). Individual values and the mean _+ SD 0 0 of each group were compared for C3, I g • I 11 III I II lII C4, and CHs0

0 -~, 2

O O O) E 2 o ol 0 0 O O o o o8 ? Y 1 -° "- 1 .12 ..... ~L.~,.L. z ~_~ Fig. 3. Serum immunoglobulin levels O ~ 08 ~ in the patients without MRSA coloni- < 2 N I o~ zation (group I), those with MRSA O ~ 8 °~I o colonization (group II), and those .~ ...... ~ ..... ~-g1 --- with MRSA infection (group III). Individual values and the mean + SD 0 of each group were compared for IgG, I II llI I 11 Ill I ]I Ill IgA, and IgM

difference was not statistically significant. The geometric Figure 4 shows the geometric values of antibody mean of IgM was 1.20 +x 0.51 in group III and 1.43 +x production for individual patients and the geometric 0.50 in groups I and II, without significant difference (P means in the three groups. The geometric mean of anti- = 0.41). PPS IgG in group III was significantly lower than that in T. Saito et al.: MRSA Infection and Defense Mechanisms 951

10000 o I0000 1000

o LLI °]~ ~~.1 W ~.~ o 1000 o ,< 1000 ~ 1oo o o "~ __~ Fig. 4. Antibody response to pneumo- coccal polysaccharide vaccine (PPS) immunization of the patients without ~_ oo ~. :o o I~. MRSA colonization (group I), those 100 o ~ 100 ;t .-- ._ o[ ~° with MRSA colonization (group II), t'- o o ~ t- 000 and those with MRSA infection (group < ,< o < o III). Individual titers and the geometric o mean 2 SD of each group were com- 0 10 10 0 pared for IgG-, IgA-, and IgM-anti- I II llI I 1I lII I II ]lI PPS/ml serum. *P < 0.05

E~ ~. 0 X o ~ 40 o I .~ O ~_~ O ~ 4 .fl ~ O~ot~ ~2 o oj. ~,0o : °] 14' 0 -r 0 0 =I ...... I II llI I g g

× 41 o ~'~~ o o ~ 4 T s × ...... ~r ...... x ? ~' i[r-~ ?...... I, 2 o g °T ~ O Fig. 5. Cell-mediated immunity in the patients ~ o o~ 8 T ~ without MRSA colonization (group I), those 82s~ Ol o~ ~ with MRSA colonization (group II), and ~ 8 2 o those with MRSA infection (group III). ':~ ...... ° ..... ;;I ~ • ...... O Individual values and the mean _+ SD were compared for the total, T, and B lymphocyte , • ~ o- ~ counts, phytohomagglutinin (PHA) transfor- ~ ~ ~ ee~ O o mation and the purified protein derivative (PPD) skin test. *P < 0.05 between group II O 0 and group III; *P < 0.05 between group I and I g • I II Ill lI IlI group II group I, being 3.26 2 0.10 (P < 0.01), and also tended lower than 600 EU who had no postoperative MRSA to be lower than that in group II of 3.00 +~ 0.70 (P = infection, surgical stress was found to have been extre- 0.08). There were no significant differences among the mely low compared to that of the other patients, the three groups for anti-PPS IgA and IgM. When the titers duration of surgery having been 4-5 h and blood loss, of anti-PPS IgG were compared among individual 160ml (Table 1). patients, they were found to be >600EU in all but As for cell-mediated immunity (CMI), the mean one of the patients who did not develop an MRSA total lymphocyte count was significantly lower in infection, whereas it was <600 EU in all of the patients patients with MRSA infections than in those without with an MRSA infection. In the patient with a titer (P < 0.05) and in group III than in group II (P < 0.05). 952 T. Saito et al.: MRSA Infection and Defense Mechanisms

Similar trends were observed for T- and B-lymphocyte infections. The total lymphocyte count and PPD skin counts, although the differences were not statistically test, but not PHA transformation, were also significantly significant (Fig. 5). The mean PPD skin test was also more reduced in patients with MRSA infection than significantly lower in group III than in group II (P < in those with MRSA colonization, although the border- 0.05). Although the mean PHA transformation was line levels of these parameters between the presence lower than the standard, there were no significant dif- and absence of MRSA infection were obscure. PMN ferences among the three groups. play an important role in the defense against Gram- Malnutrition was observed in half the patients, positive cocci such as Staphylococcus aureus. However, although there were no significant differences in the an enhancement in SOP and MPO and a reduction eight nutritional parameters among the three groups in the KI of PMN were not associated with MRSA (data not shown). Correlations between infection or infection. The enhanced levels of complements such as colonization with microorganisms other than MRSA C4 and CHs0, and of the polyclonal immunoglobulins and impairment of the host defense mechanisms were such as IgA were also unrelated. Thus, it seems analyzed but no significant differences were seen. apparent that a deficiency in the antibody-producing capacity, with a partial deficiency in CMI is associated with MRSA infection following esophageal surgery. The antibody response to various antigens are dif- Discussion ferentially dependent on T cells, 26 while the response to protein antigens such as tetanus toxoid is classi- In our hospital, the outbreak of MRSA was most cally considered to require the participation of T prevalent at the time of this study, when the highest cells. 27 Pneumococcal polysaccharide which was used incidence of infected or colonized patients and staff was in the present study, is a relatively T-cell-independent recorded. The isolation of MRSA was characterized by bacterial antigen, a8 However, the deficiency in the type II-coagulase, type A or C enterotoxins, and a antibody-producing capacity may partly depend on the positive production of the toxic shock syndrome toxin- impairment of T-cell immunity, which was also related 1. MRSA, though not evident prior to surgery, was to the development of MRSA infections. isolated in the oropharyngeal swabs on postoperative The present finding is important because it suggests days 3 to 4, then in the tracheal aspirate on day 7 or that patients with a reduced capacity of anti-PPS later in patients with esophageal cancer (unpublished IgG production on admission are at a high risk of data). MRSA was therefore spread by the hospital developing MRSA infection following esophageal staff, or air borne in the surgical ICU or surgical surgery if the pathogens are colonized. Conversely, ward. The most common portals of entry for MRSA patients with a normal ability to produce anti-PPS IgG pneumonia seemed to be the respiratory tract, occurring will probably not develop an MRSA infection even if through intubation or tracheostomy for mechanical colonization occurs. Thus, the preoperative evaluation ventilation. of anti-PPS IgG production seems useful for predicting In the present study, we studied the relationship the risk of MRSA infection following major surgery between the preoperative impairment of host defense such as esophagectomy. Moreover, the anti-PPS IgG mechanisms and post-operative MRSA infection fol- titer of 600EU, may be the critical value in the pre- lowing esophageal surgery. The anti-PPS IgG antibody diction of a postoperative MRSA infection. response was significantly lower in patients with post- Many risk factors are related to the operative mor- operative MRSA infections than in those without. bidity and mortality following transthoracic esophagec- Furthermore, the titer of anti-PPS IgG was >600 EU in tomy. ~9 Preoperative risks include old age, underlying all the MRSA-infected patients and <600EU in all diseases, disorders of the systemic vital organs, mal- but one of the non-infected patients. These findings nutrition, and the stage of malignancy. Surgical stress indicate that the reduced capacity of anti-PPS IgG factors, such as intraoperative blood loss and the production prior to surgery is associated with the duration of surgery, and postoperative management, development of MRSA infection following esophageal including nutritional and cardiopulmonary care, are surgery. The finding that the anti-PPS IgG response also important. The factors associated with the acquisi- was similar in patients with MRSA colonization and tion of MRSA 6'8'9 include the interval between admis- those without also suggested that impaired antibody- sion and the development of MRSA, the administration producing capacity is not related to colonization with of antibiotics, and care in the surgical ICU, such MRSA. as mechanical ventilation through endotracheal intu- Among the preoperative impairment of other com- bation, and intravenous hyperalimentation. In the ponents of the host defense mechanisms, a deficiency in present study, none of these factors significantly dif- CMI was partly associated with postoperative MRSA fered among the patients with MRSA infections, those T. Saito et al.: MRSA Infection and Defense Mechanisms 953

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