methicillin-resistantstaphylococcus aureus proliferation in the rat gut is influenced by gastric...

327Y. Yoshida: MRSA Proliferation in Rat GutSurg TodayJpn J Surg (1999) 29:327–337

Reprint requests to: Y. Yoshida(Received for publication on Feb. 2, 1998; accepted on Nov. 6,1998)

fatal. It has been suggested that various factors, suchas the administration of the third-generation cepha-losporin group of antibiotics,1–3 upper gastroenter-ological surgery,1 and gastric acid inhibition due to theadministration of histaminergic H2 receptor antagonists(H2RA),2–7 may be involved in the development ofMRSA enterocolitis. It is therefore of interest to exam-ine the influence of H2RA and antimicrobial agentson the intestinal MRSA proliferation in experimentalmodels.

Materials and Methods

Animals

Sixty-five male rats of Wistar/ST strain (Japan SLC,Shizuoka, Japan) with a body weight of around 250 gwere used. The rats were kept in metabolic cages, andwere fed with tap water and solid rat chow CE-2 (CleaJapan, Tokyo, Japan) freely for 1 week to acclimatize tothe living conditions, then were used for the followingexperiments.

Separation of Bacteria

The rats were anesthetized by an intravenous injectionof pentobarbital diluted 10 times with physiological sa-line and were immediately subjected to the operation.Their stomaches, small intestines (from Treiz’s suspen-sory ligament to 20 cm anal side), and large intestineswere removed aseptically, and stored in an anaerobicporter (Clinical Supply, Gifu, Japan). The contentsof each intestine were diluted 10 times using an anaero-bic bacterial diluting solution,8 whose atmospherewas replaced by CO2 gas in an anaerobic chamber,composed of 4.5 g KH2PO4, 6.0g Na2HPO4, 0.5g l-cysteine · HCl ·H2O, 0.5g polyoxyethylene (20) sorbitanmonooleate (Wako Junyaku Kogyo, Osaka, Japan),1.0 g nutrient agar (Eiken Chemical, Tokyo, Japan), and

Abstract: The author studied methicillin-resistant Staphylo-coccus aureus (MRSA) proliferation in the rat gut which wasinfluenced by gastric acid inhibition and the administrationof antibiotics. When male Wistar rats were bred by totalparenteral nutrition (TPN), and were continuously adminis-tered famotidine 4 mg/kg per day, the gastric acidity was ob-served to decrease to pH 6.4 6 0.1. However, when they werebred by TPN, and histamine 4 mg/kg per hour was continu-ously administered, the gastric acidity was observed to in-crease to pH 1.9 6 0.4. MRSA was thus able to cross over tothe small intestine only during the famotidine medication. Ifrats were intravenously administered latamoxef (LMOX)after an oral inoculation of MRSA, then the viable MRSAcounts in the stomach, small intestine, and large intestine alldecreased on day 4. In contrast, if the gastric acidity decreasedand the rats were treated by an oral administration of kanamy-cin and metronidazole before an oral inoculation of MRSAand thereafter were administered LMOX, then the MRSAcount significantly increased. It is thus concluded that a sup-pression of gastric acid and a great disorder of the intestinalflora is indispensable for the colonization of MRSA intothe small intestine, while in vitro the propagation of MRSArequires a continuity of suppression absent in the bacterialflora.

Key Words: methicillin-resistant Staphylococcus aureus,histamine H2 antagonist, gastric acid, antibacterial colonpreparation

Introduction

Enterocolitis due to methicillin-resistant Staphylococ-cus aureus (MRSA) is one of the serious postoperativecomplications which requires utmost care because if thestart of treatment is late, the outcome can sometimes be

Methicillin-Resistant Staphylococcus aureus Proliferation in theRat Gut is Influenced by Gastric Acid Inhibition and theAdministration of Antibiotics

Yuichi Yoshida

Third Department of Surgery, Toho University School of Medicine, 2-17-6 Meguro-Ward, Tokyo 153-8515, Japan

328 Y. Yoshida: MRSA Proliferation in Rat Gut

1000ml pure water, and these suspensions were thenspread onto agar plates.

For the culture media for measuring total aerobicbacterial numbers, blood agar (5% defibrinated horseblood was added) plates were used. DHL agar (NissuiPharmaceutical, Tokyo, Japan) plates were used toseparate the Escherichia coli, while mannitol salt cul-ture agar (Eiken Chemical, Tokyo, Japan) plates wereused to separate the Staphylococcus species, MuellerHinton agar (Difco Laboratories, Detroit, MI, USA)plates containing 4µg/ml of oxacillin (MPIPC; titer,845mg/mg; Banyu Pharmaceutical, Tokyo, Japan) and4% of NaCl were used to separate MRSA, and EF agar(Nissui Pharmaceutical) plates were used to separateEnterococcus faecalis. To measure the total viable num-ber of anaerobic bacteria, GAM agar (Eiken Chemical)plates were used. BBE agar (Kyokuto Pharmaceutical,Tokyo, Japan) plates were used to separate theBacteroides species.

Bacterial Culture and the Method of Identification

For detecting the aerobic bacteria, the plates were culti-vated at 35°C for 48h. The colonies were identified byGram staining, the oxidase test, the catalase test, andthe VITEK method. At this time, Gram-positive bacte-ria, which appeared on the mannitol-salt agar plates,were subjected to the coagulase test. As for the EF agarplates, after cultivation for 48h, the plates were kept atroom temperature for 24h and then the colonies wereassessed.

To detect the anaerobic bacteria, plates were culti-vated in an anaerobic culture apparatus (Model 1024,Anaerobic System; Forma Scientific, OH, USA) at 35°Cfor 48 h, and after Gram staining, the indol test wasconducted and identification was performed using theVITEK method.

Measurement of pH

In parallel with the bacterial investigation in the rats,the pH of the gastric juice, small intestinal juice, andlarge intestinal juice was measured using a pH meter(type pH BOY-p1, Shin Dengen Kogyo, Tokyo, Japan).

Experimental Groups

Preparation of MRSA Noninoculated GroupA central venous line was inserted into the rats, the ratswere abstained from food, and the following experi-mental models were prepared by total parenteral nutri-tion (TPN).

Group of Oral Nutrition (ON Group). A group of ratsfed with oral nutrition for 1 week (n 5 6).

TPN Group. Rats of the ON group were fastened, ajugular vein catheter was inserted according to themethod of Steiger et al.,9 and TPN was conducted (n 511). From the starting day until the first day, 2.20ml/h/body ·15.84 kcal/day of Amicaliq (Terumo, Tokyo,Japan), until the fourth day, 2.57ml/h/body · 46.60kcal/day (600 ml of Triparen-1 (Otsuka Pharmaceutical, To-kyo, Japan), 300 ml of Amiparen (Otsuka) and OtsukaMV injection set (Otsuka) were mixed, the mixture wasdivided into 300ml and packaged aseptically into aKawasumi Total Parenteral Nutrition Bag H-10J(Kawasumi Laboratories, Tokyo, Japan)), until the fifthday, 2.67ml/h/body · 58.38 kcal/day, and from the sixthday, 3.00ml/h/body ·65.60 kcal/day (600ml of Triparen-2 (Otsuka), 300ml of Amiparen, and Otsuka MV injec-tion set were mixed) were administered.

Group of TPN 1 H2RA Continuous Infusion (TPN 1H2RA Group). A group of rats in the TPN group inwhom TPN was continuously instilled with the additionof 4mg/kg per day of famotidine (titer; 1000 µg/mg,Yamanouchi Pharmaceutical, Tokyo, Japan) for 24h(n 5 12).

Group of TPN and Histamine Continuous Infusion(TPN 1 Hist Group). A group of rats was continuouslyinstilled with 4mg/kg per hour of histamine (titer morethan 95%, Wako Junyaku) (n 5 9) as a gastric acidsecretion stimulant. Histamine was dissolved immedi-ately before use in the instillion bag containing the in-stillation volume for 1 day. The administration quantityof histamine was determined based on the histamineadministration experiments of gastric fistula rats.10–14

Preparation of MRSA-Inoculated GroupUsing models of various gastric acid secretion condi-tions prepared according to the protocol outlinedabove, MRSA oral inoculation experiments wereperformed.

The bacterium used was the MRSA328GTS strain,which was separated from an MRSA enterocolitis pa-tient and preserved in our school laboratory in a frozencondition, and after cultivation for 24h by mannitol saltplates containing 4µg/ml of MPIPC, the bacteria weretransferred to Mueller Hinton agar containing 32µg/mlof latamoxef (LMOX; titer, 912µg/mg; Shionogi,Osaka, Japan), and cultivated for 24h, then cultivatedon blood agar plates statically for 18h. This was thendiluted with sterilized physiological saline and wasmade into a solution of living bacteria of 5.0 3 109

colony-forming units (cfu)/ml using a spectrophoto-meter (Model 220A, Hitachi, Tokyo, Japan).

This bacterial solution was inoculated in rats at abacterial quantity of 109 cfu/body orally, using a stom-ach sounder on the fourth day from the start of TPN,

329Y. Yoshida: MRSA Proliferation in Rat Gut

and the following experimental animal groups wereprepared. Each rat was killed on the eighth day afterstarting TPN (fourth day of inoculation of MRSA)and then the bacterial investigation was conducted.TPN 1 H2RA 1 MRSA group: TPN 1 H2RAgroup to which MRSA was orally inoculated (n 5 3);TPN 1 MRSA group: TPN group to which MRSAwas orally inoculated (n 5 3); TPN 1 hist 1 MRSAgroup: TPN 1 hist group to which MRSA was orallyinoculated (n 5 3).

Preparation of Group Administered AntibioticThe oral inoculation of MRSA was made on the fourthday after starting TPN, and the administration ofLMOX was started from the day of inoculation. Eightymg/kg per day of LMOX was dissolved in 1 ml of physi-ological saline, and it was then instilled intravenouslyonce a day while taking more than 30min through theside line of a three-way valve affixed to the catheter.LMOX was preliminarily administered at a concentra-tion of 20 mg/kg per day to rats (n 5 3), but no signifi-cant changes were observed in the viable bacterial countof E. coli obtained from the intestinal bacterial floraafter the treatment. Arima,15 who developed MRSAcarrier rats, and Aoyagi,16 who administered LMOXwith other antibiotics to rats along with MRSA inocula-tion, used LMOX at a concentration of 80 mg/kg perday, and they obtained fairly good results. For thesereasons, I also used LMOX at a concentration of 80mg/kg per day in this study.

The following three groups were prepared: TPN 1H2RA 1 MRSA 1 LMOX group: TPN 1 H2RA groupto which MRSA was orally inoculated on the fourth dayafter starting TPN, and therefore LMOX was instilledintravenously once a day continuously (n 5 3); TPN 1MRSA 1 LMOX group: TPN group to which MRSAwas orally inoculated on the fourth day after startingTPN, and then LMOX was instilled intravenously oncea day continuously (n 5 3); TPN 1 hist 1 MRSA 1LMOX group: TPN 1 hist group to which MRSA wasorally inoculated on the fourth day after starting TPN,and then LMOX was instilled intravenously once a day(n 5 3).

Next, using the TPN 1 H2RA group, which was agastric acid secretion depressed model, pretreatmentwith antibacterial colon preparation (ACP) was madeon the rats before the oral inoculation of MRSA.

ACP was carried out by a suspension in 0.5% CMC(carboxymethyl cellulose sodium salt (Wako Junyaku);after sterilization at 121°C for 15 min, the suspensionwas cooled down to room temperature and then used),to which 100 mg/kg per day of kanamycin (KM;titer, 690 µg/mg; Meiji Seika Kaisha, Tokyo, Japan) and25 mg/kg per day of metronidazole (MTN; titer,1000 µg/mg; Shionogi) were mixed in (KM 1 MTN).

ACP Group. The TPN 1 H2RA group of rats to whichACP was carried out by KM 1 MTN for 4 days, andafter killing the rats, a bacterial investigation was per-formed (n 5 3). Subsequently, MRSA was inoculatedorally to the ACP group.

ACP 1 MRSA Group. The TPN 1 H2RA group ofrats to which ACP was carried out by KM 1 MTN for 4days, and MRSA was orally inoculated, and on thefourth day (eighth day after starting TPN) the ratswere killed and a bacterial investigation performed (n 53). Furthermore, third-generation cephalosporin anti-biotics were administered to the ACP 1 MRSA group.

ACP 1 MRSA 5 LMOX Group. The TPN 1 H2RAgroup of rats to which ACP was carried out by KM 1MTN for 4 days, and MRSA was orally inoculated, and80 mg/kg per day of LMOX was instilled intravenouslyonce a day for more than 30 min for 4 days (n 5 3).

Statistical Analysis and Evaluation

The number of living bacteria (cfu/ml) in each experi-mental group was examined for compatibility, andbecause a normal logarithmic distribution was found, itwas evaluated after calculating the log(cfu/ml) value.For evaluations between the two groups, an F examina-tion was conducted at first, and if no difference wasfound between the two groups by the F examination,then the t-test was conducted. If there was a differencein the F examination, the Aspin-Welch examinationwas conducted. For multi-group evaluations, afterBartlett’s statistic was calculated the F examination wasconducted, and after error processing, the experimentswere performed and an examination of the difference inthe average values (multiple comparison) for each levelwas conducted. The level of significance of the analysiswas set at 5%.

Results

Microbial Flora in Non-MRSA-Ingested Groups

In the ON group, the pH of alimentary juice was 4.18 60.52 in the stomach, 6.02 6 0.26 in the small intestine,and 6.25 6 0.38 in the large intestine.

The total amount of aerobes in the stomach was107.54 cfu/ml including 103.71 cfu/ml of E. coli, 102.84 cfu/mlof S. aureus, and 103.96 cfu/ml of E. faecalis (Fig. 1). Theamount of anaerobes totaled 109.57 cfu/ml, and colonyformation of Bacteroides fragilis was not observedbeyond the limit of the detection assay.

The total amount of aerobes in the small intestineas 107.01 cfu/ml including 102.82 cfu/ml of E. coli and102.40 cfu/ml of S. aureus. E. faecalis was not detected by


Fig. 1. Microbial flora in the ON (oral nutrition) group

Fig. 2. Gastrointestinal acidity of rats managed with totalparenteral nutrition (TPN). Squares, stomach; circles, je-junum; triangles, colon

Fig. 4. Changes in the jejunal microbial flora of rats managedwith TPN

Fig. 3. Changes in the gastric microbial flora of rats managedwith TPN

the detection assay. The total amount of anaerobes was107.98 cfu/ml. No colonies of B. fragilis were observed.

The total amount of aerobes in the large intestine was108.19 cfu/ml including 106.81 cfu/ml of E. coli, 104.83 cfu/mlof S. aureus, and 106.10 cfu/ml of E. faecalis. The totalamount of anaerobes in the organs was 109.53 cfu/mlincluding 106.08 cfu/ml of B. fragilis.

No significant difference in the pH of the digestivejuice in the stomach and the small or large intestine wasobserved in the TPN group (Fig. 2).

The amount of microbes in the stomach decreasedremarkably 2 days after the start of TPN. 104.54 cfu/ml ofaerobes and 105.10 cfu/ml of anaerobes could be detected4 days after the start of feeding (Fig. 3). The amount ofS. aureus was at the detection limit of the assay, and nosignificant difference in the amount of the microbes wasobserved before or after the start of the feeding.

The total amounts of aerobes and anaerobes in thesmall intestine were not significantly different eitherbefore and after TPN feeding. The amount of E. coliobtained 2 days after the TPN feeding (approximately105 cfu/ml) outnumbered that observed before the TPNfeeding (Fig. 4). In addition, numbers of cfu of S. aureus,E. faecalis, and B. fragilis were all below 104 per ml afterTPN feeding.

The abount of E. coli in the large intestine increasedby 109 to 1010 cfu/ml 2 days after the TPN feeding. Theamount of E. faecalis also increased by 108 cfu/ml 6 daysafter TPN feeding (Fig. 5). However, no significant dif-ference in the amount of S. aureus was recognized be-tween that before and after the start of the TPN feeding.The amount of feces obtained from rats decreased re-markably a few days after the start of the TPN feedingand was 0.005 to 0.061g per rat 6 days thereafter.

In the TPN 1 H2RA group, the pH of gastric juicesignificantly increased after 2 days of the treatment andreached 6.40 6 0.10 6 days after the treatment. The pH


Fig. 5. Changes in the colonic microbial flora of rats managedwith TPN

Fig. 6. Gastrointestinal acidity of rats managed with TPNsupplemented with histaminergic H2-receptor antagonist(H2RA; famotidine). Squares, stomach; circles, jejunum;triangles, colon

of small and large intestinal juice was between 6 and 7,respectively (Fig. 6).

The total amount of microbes in the stomach de-creased remarkably after 2 days of the treatment (Fig.7). The amount of bacteria obtained 4 days after thetreatment as larger than that obtained on the same dayin the TPN group, showing 106.57 cfu/ml in aerobes and106.11 cfu/ml in anaerobes.

The amounts of E. coli, S. aureus, and E. faecalis inthe small intestine increased 2 to 4 days after the treat-ment, although the total number of cfu of aerobes oranaerobes was not significantly different during theperiod (Fig. 8). The amount of aerobes decreased by105.04 cfu/ml, and that of anaerobes by 105.09 cfu/ml, 6 daysafter the treatment. In addition, the amounts of E. coli,S. aureus, and E. faecalis also decreased significantly.

Fig. 7. Changes in the gastric microbial flora of rats managedwith TPN 1 H2RA

Fig. 8. Changes in the jejunal microbial flora of rats managedwith TPN 1 H2RA

The amounts of E. coli and E. faecalis in the largeintestine increased by 109 to 109.5 cfu/ml and 108 cfu/ml,respectively (Fig. 9). The amount of S. aureus did notchange significantly 2 days after the treatment.

In group TPN 1 hist, the pH of the gastric juicebecame strongly acidic 2 days after the treatment andindicated 1.87 6 0.38 after 6 days of the treatment(Fig. 10).

No bacteria were isolated from the stomach in thegroup on any days after the second day of the treatment(Fig. 11). The amount of microbes in the small intestinedecreased by less than 104 cfu/ml 6 days after the startof the treatment (Fig. 12). The cfu/ml of E. coli andE. faecalis in the large intestine increased by approxi-mately 1010.5 and 109, respectively, after the second dayof treatment (Fig. 13). However, the amount of S.aureus did not change significantly throughout theexperiment.


Fig. 9. Changes in the colonic microbial flora of rats managedwith TPN 1 H2RA

Fig. 10. Gastrointestinal acidity of rats managed with TPNsupplemented with histamine (hist). Squares, stomach; circles,jejunum; triangles, colon

Fig. 11. Changes in the gastric microbial flora of rats managedwith TPN 1 hist

Fig. 12. Changes in the jejunal microbial flora of rats managedwith TPN 1 hist

Fig. 13. Changes in the colonic microbial flora of ratsmanaged with TPN 1 hist

Microbial Flora in MRSA-Inoculated Groups

In the TPN 1 H2RA 1 MRSA group, the number ofcfu/ml of MRSA was 105.05 in the stomach, 105.30 in thesmall intestine, and 105.42 in the large intestine. How-ever, the amounts of the microbes other than MRSAobtained from the small intestine as well as from thestomach were also larger as the pH of the gastric juiceapproached neutrality (Fig. 14).

No colony-forming MRSA was observed in thesamples obtained from the digestive tract in either theTPN 1 MRSA group or the TPA 1 hist 1 MRSAgroup with the exception of the stomach in the TPN 1MRSA group, thus showing that MRSA could notinvade the small or large intestine in the groups whosegastric juice did not have low pH levels.

Microbial Flora in Antibiotic-Administered Groups

MRSA was not detected from the alimentary tracts ofany TPN 1 H2RA 1 MRSA 1 LMOX, TPN 1 MRSA


Fig. 14. Comparison of microbial flora in the methicillin-resis-tant Staphylococcus aureus (MRSA) inoculated groups

1 LMOX, or TPN 1 hist 1 MRSA 1 LMOX groupsbeyond a lower limit of the assay (Fig. 15).

Neither aerobic nor anaerobic bacteria were detectedfrom any digestive tracts pretreated with antibiotics inthe ACP group (Fig. 16).

Fig. 15. Comparison of microbial flora in groups administeredlatamoxef (LMOX) after MRSA inoculation

The amount of MRSA in the ACP 1 MRSA groupwas 105.13 cfu/ml in the stomach, 104.74 cfu/ml in the smallintestine, and 105.36 cfu/ml in the large intestine. 105.13 cfu/ml of E. coli and 103.79 of E. faecalis were also detected


administered groups, 107.07 cfu/ml in the stomach,105.91 cfu/ml in the small intestine, and 106.80 cfu/ml in thelarge intestine, although the cfu/ml of E. coli was notdetected beyond the lower limit of the assay.

Discussion and Conclusions

The incidence of MRSA enteritis has increased rapidly,in Japan since the mid-1980s.1 The disease has been paidmuch attention by most communities, because the de-layed diagnosis of the patients who undergo laparotomymay cause sepsis, shock,17 and multiple organ failure,18

and also result in nosocomial infection. Fundamentalstudies about the onset of MRSA enteritis have yet tobe carried out, although several causes such as uppergastrointestinal tract surgery, administration of H2RA,the administration of third-generation cephalosporingroup antibiotics,1–3 gastric acid inhibition due to H2RAadministration,2,7 and superinfection,19 have beenproposed. I therefore investigated the growth rate ofMRSA in digestive organs of rats in which the pH ofgastric juice was increased by H2RA.

The rats used in the study were fed in the cages inwhich their feces could be obtained from the outside.This is because the rats fed in regular cages sometimesshow coprophagia, and B. fragilis is also sometimesisolated from such rats. Coprophagia seen in rats is abehavior by which the rats attempt to ingest nutritionalrare elements that are not easily absorbed from thedigestive tract, especially iron. It is said that rats pro-tected against coprophagia tend to develop anemiaafter several months. The rats used in this study, how-ever, did not develop either element insufficiency oranemia, since adequate amounts of those rare elementswere administer to them. I believe that rats should bekept in metabolic cages when performing experimentssimilar to those described above, because humans nevershow coprophagic behavior.

The amounts of microbes in the ON group with gas-tric juice of pH 3–4 were larger than those in the TPN 1H2RA group with a fairly raised pH in the gastric juiceby H2RA. In addition, the amounts of microbes in theTPN group were smaller than those in the TPN 1H2RA group. These data suggest that food debris in thestomach could be a substitute for peptone agar for bac-teria. The gradual decrease in the amount of anaerobesisolated from the stomach and jejunum in the TPNgroups appears to be related to parenteral nutrition. Ialso observed, through the data from the TPN 1 histgroup, that rats were capable of secreting high concen-trations of hydrogen chloride into the stomach.

MRSA could not survive in the stomach under thehighly acidic conditions found in such cases as in theTPN 1 hist group with hypersecretion of gastric acid.

in the group. No significant difference in the totalamount of MRSA was observed between the ACP 1MRSA and TPN 1 H2RA 1 MRSA groups.

The amount of MRSA in the ACP 1 MRSA 1LMOX group was the largest of all the antibiotic-

Fig. 16. Comparison of microbial flora in groups to which anantibacterial colon preparation was added prior to the MRSAinoculation


Since MRSA could not be detected in any digestiveorgans apart from the stomach in the TPN group, itseems that MRSA cannot invade the small and largeintestines unless the pH of gastric juice is intentionallyincreased. Kawai studied the growth and survival ratesof MRSA in rats with a resected stomach in comparisonwith those in stomach-nonresected rats, by pouringMRSA suspensions into their duodenum.20 Okada et al.reported that although patients with a perforation of agastroduodenal ulcer were administered H2RA, noMRSA enteritis was observed.21 Miyake et al. investi-gated the etiology of MRSA enteritis and reportedthat there was no significant difference in the onsetof MRSA enteritis between the patients with agastric resection or administration of H2RA and thecontrol group.22 Suzuki obtained gastric juice throughtransnasal gastric tubes and found that MRSA could notbe isolated from the juice after patients were givenhydrochloric lemonade drinks.4 He also reported thatif patients were not given hydrochloric lemonadedrinks and MRSA could be isolated from their gastricjuice, they did not develop MRSA enteritis.4 AlthoughMRSA is not said to be isolated from the feces of pa-tients affected with MRSA in their respiratory tract,23

Itoh et al. showed that patients suffering from chronicrespiratory disease showed an 85.2% MRSA-positiverate in their feces.24 However, it is unclear as to whetherthey developed enteritis due to an overproliferation ofMRSA in their digestive organs, since most of them hada solid stool.

The invasion of MRSA into alimentary tracts is thusnot sufficient for the pathogen to be colonized in thesetracts. The present study, in which the rats in the TPN 1H2RA group were infected with MRSA, revealed theamounts of E. coli and E. faecalis to be larger than thatof MRSA, thus indicating that the carrier state in aninfection with MRSA does not always lead to an over-growth of the pathogen in the alimentary tracts.

Reports on MRSA enteritis have been increasingsince the use of third-generation cephalosporins has in-creased in postoperative patients. Therefore, many phy-sicians now know that third-generation cephalosporinsfacilitate the production of penicillin-binding protein(PBP)-29, because MRSA strains can grow in the pres-ence of â-lactam antibiotics because of the low affinitiesof the specific PBP29 fraction for various â-lactamantibiotics.25 However, few researchers think thatMRSA, which does not have a mecA gene, wouldnever be mutated by the cephalosporins and thus be-came resistant,26 since methicillin resistance in staphylo-cocci is primarily due to the presence of the mecA genewhich encodes the novel PBP 29.27 Most physicians be-lieve that the third generation of cephalosporins sup-press the proliferation of Gram-negative bacteria andMRSA.28

Kawai reported in his previous study that when ratswith a resected stomach were given MRSA and LMOX,the amount of Gram-negative bacilli in their feces rap-idly decreased 4 days after the start of the treatment and104 cfu/ml of MRSA were isolated from the feces.20 Inthe present study, no overgrowth of MRSA was foundin rats even if LMOX was given for 4 days after themicrobe had been orally introduced into them and hadalso invaded the small intestine. As a result, factorsother than the pH of gastric juice appear to be involvedin the invasive proliferation of MRSA in the digestiveorgans.

Aoyagi studied the in vitro effects of antibiotics onthe proliferation of MRSA by coculturing GAM brothwith E. coli, E. faecalis, and B. fragilis, and revealed theamount of MRSA after coculturing to be smaller thanbefore treatment, when one of the three species weresuppressed in growth by antibiotics and that the amountof MRSA after coculturing was larger than before treat-ment when the growth of more than one of the otherspecies than MRSA was inhibited by antibiotics.16 Wealso had similar in vivo results in the CP group; MRSAcould not be isolated from patients who were pretreatedwith KM 1 MTN before MRSA was given orally but105 cfu/ml of MRSA were detected in the CP 1 MRSAgroup.

However, treatment with KM 1 MTN, which sup-presses the proliferation of more than one species ofbacteria, is not sufficient for the onset of MRSAenteritis, because there is no significant difference inthe amount of MRSA between the CP 1 MRSA groupand the TPN 1 H2RA 1 MRSA group, and similaramounts of E. coli and E. faecalis were detected in bothgroups. This is because E. coli and E. faecalis grow andincrease in number after the inhibitory effect of anti-biotics on the proliferation of microbes disappears.

The overgrowth of MRSA is most likely induced inthe alimentary tract, when antibiotics are given afterpatients are pretreated with an antibacterial colonpreparation before surgery. In the present study, 107 cfu/ml of MRSA in the CP 1 MRSA 1 LMOX group wasthe largest amount obtained after treatment in all thegroups, probably because the rats in this group had beentreated with CP and the growth of E. coli was thusinhibited. MRSA appears to grow best under such con-ditions. 107.07 cfu/ml of MRSA isolated from the stomachappears to be about the maximal level of cfu with whichMRSA can be isolated from any part of the alimentarytract.

The reason why larger amounts of MRSA were iso-lated from the stomach than the jejunum, which is amain focus of MRSA infection, is partly because therats in this group exhibited diarrhea. In other words,the jejunum infected with MRSA produced so muchdigestive juice that the amount of microbes became


relatively small (105.91 cfu/ml) because of dilution withjejunal juice.

Okamura et al. reported that patients with coloniccancer who had been pretreated with 2g of KM for4 days before surgery, and had been given H2RAimmediately after surgery, developed MRSA enteritis.29

These results suggest that the postoperative use of anti-biotics plays a role in the onset of MRSA enteritis.

These data also suggest that the neutralization ofgastric juice brought by gastric resection and theadministration of antacids allows MRSA to invadelower digestive organs. In addition, pretreatment withan antibacterial colon preparation before surgery is alsonecessary for MRSA to invade lower digestive organs.When antibiotics such as LMOX are given after theinvasion of MRSA into the digestive tracts and growthof other bacteria is inhibited, MRSA enteritis may thusdevelop. Since patients who undergo abdominal surgeryneed to be given antibiotics postoperatively, surgeonsshould not overuse antibiotics before surgery, andshould also try to use only a minimal amount of antibi-otics after surgery, in order to prevent MRSA enteritis.

Acknowledgments. I would like to express my sincere grati-tude to Prof. Yoshinobu Sumiyama of the Third Departmentof Surgery and to Prof. Keizo Yamaguchi of the Departmentof Microbiology of the Toho University School of Medicinefor their critical reading of the manuscript and valuablesuggestions. I also would like to thank Assistant Prof. ShinyaKusachi of the Third Department of Surgery and AssistantProf. Shuichi Miyazaki of the Department of Microbiologyof the Toho University School of Medicine for their guidancethroughout this study, and the staff members of the ThirdDepartment of Surgery and the Department of Microbiologyof the School for their cooperation.

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methicillin-resistantstaphylococcus aureus proliferation in the rat gut is influenced by gastric...

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