JOURNAL OF CLINICAL MICROBIOLOGY,0095-1137/98/$04.0010

Oct. 1998, p. 3081–3084 Vol. 36, No. 10

Copyright © 1998, American Society for Microbiology. All Rights Reserved.

Genetic Relationship between Blood and Nonblood Isolatesfrom Bacteremic Patients Determined by Pulsed-Field

Gel ElectrophoresisJUNICHI MATSUDA,1 YOICHI HIRAKATA,1* FUMIAKI IORI,1 CHIKAKO MOCHIDA,1 YUMI OZAKI,1

MICHIKO NAKANO,1 KOHICHI IZUMIKAWA,1 TOSHIYUKI YAMAGUCHI,1 RYOJI YOSHIDA,1

YOSHITSUGU MIYAZAKI,1 SHIGEFUMI MAESAKI,2 KAZUNORI TOMONO,2

YASUAKI YAMADA,1 SHIGERU KOHNO,2 AND SHIMERU KAMIHIRA1

Department of Laboratory Medicine,1 and Second Department of Internal Medicine,2

Nagasaki University School of Medicine, Nagasaki 852-8501, Japan

Received 31 March 1998/Returned for modification 6 June 1998/Accepted 1 July 1998

A total of 148 isolates from 55 bacteremic patients were examined by pulsed-field gel electrophoresis.Genetically different nonblood strains were isolated from 13.9% of patients with bacteremia caused by gram-positive cocci and 42.1% with Pseudomonas aeruginosa bacteremia, indicating that antibiograms of a singlenonblood P. aeruginosa isolate are not always informative for treatment of bacteremia.

Bacteremia arises from preexisting local infections such aspneumonia, bile tract infection, and wounds in some patients,while in immunocompromised patients, it frequently arises as aresult of invasion by endogenous microflora (1, 9, 15). More-over, there are bacteremias with no identifiable source, such asoccult bacteremia of childhood. For patients with leukemia,surveillance culture of stool or throat samples is clinically im-portant since primary bacteremia frequently occurs during che-motherapy through colonization of the intestinal tract or throat(8, 15). In patients with primary lung cancer, infections, includ-ing endogenous bacteremia, also occur frequently during che-motherapy (5, 7). If strains isolated from nonblood clinicalspecimens prior to acquisition of a positive blood culture areidentical to blood isolates, antibiograms and other informationshould be useful in the prophylaxis and treatment of bactere-mia. However, it remains to be clarified whether blood isolatesare genetically identical to isolates from nonblood specimens(6, 11). Nor is it clear whether there are any differences be-tween blood and other isolates among bacterial species. Toaddress these points, we examined genetic relationships be-tween clinical blood isolates and nonblood isolates of the samespecies of bacteria by pulsed-field gel electrophoresis (PFGE).

The study was carried out in Nagasaki University Hospital,an 829-bed hospital in Nagasaki, Japan. Freeze-dried stockedclinical strains isolated between January 1992 and December1996 were used. Clinical isolates were identified by VitekGram-Positive and -Negative Identification cards (bioMerieux-Vitek, Inc., Hazelwood, Mo.). Staphylococci were also identi-fied simultaneously with an ID 32 STAPH kit (bioMerieux Sa,Marcy l’Etoile, France), and streptococci and enterococci wereidentified with a rapid ID 32 STREP kit (bioMerieux). Patientswith episodes of positive blood culture associated with isola-tion of the same bacterial species from other clinical specimenswithin a month before positive blood culture was obtainedwere consecutively enrolled in this study. A septic episode ofbacteremia was defined as described previously (2, 13), i.e., asfollows: (i) the first positive culture or (ii) a new positive blood

culture occurring 48 h after the preceding positive culture. Thenumbers of cases and isolates tested for each bacterial speciesare listed in Table 1. In total, 148 isolates, including methicil-lin-susceptible Staphylococcus aureus (MSSA), methicillin-re-sistant S. aureus (MRSA), coagulase-negative staphylococci(CoNS), Streptococcus pneumoniae, enterococci, and Pseudo-monas aeruginosa, from 55 patients were examined.

Chromosomal DNA was prepared from each strain and di-gested with restriction enzymes by using GenePath group re-agent kits (Bio-Rad Laboratories, Hercules, Calif.) except forS. pneumoniae isolates. SmaI was used for staphylococci andenterococci, while SpeI was used for P. aeruginosa in accor-dance with the manufacturer’s instructions. The samples wereelectrophoresed with the Gene Navigator system (PharmaciaLKB Biotechnology, Uppsala, Sweden) at 170 V for 24 h, withpulse times ranging from 5 to 60 s for staphylococci and en-terococci, and at 180 V for 22 h, with pulse times ranging from10 to 45 s for P. aeruginosa (10). Genomic DNA of S. pneu-moniae was prepared, digested with SmaI, and processed forPFGE as described previously (4, 14). Electrophoresis wascarried out at 200 V for 20 h, with a pulse time of 9.5 s. Alambda DNA ladder (Bio-Rad) was used for each PFGE as amolecular size marker. Thereafter, gels were stained withethidium bromide before being photographed under UV trans-illumination. The criteria reported by F. C. Tenover et al. (12)were applied to the interpretation of the DNA restriction pat-terns produced by PFGE. Briefly, each strain was classified asindistinguishable, closely related, possibly related, or differentif the number(s) of fragment differences compared with a ref-erence strain was 0, 1 to 3, 4 to 6, or $7, respectively. If thestrains were indistinguishable, closely related, or probably re-lated on the basis of DNA restriction patterns, they were con-sidered derivatives from a common ancestor.

Figure 1 shows PFGE patterns of MRSA strains from pa-tients with bacteremic episodes. All PFGE profiles of non-blood isolates were identical to those of blood isolates forpatients A through D. Although an isolate from the throat wasidentical to a blood isolate from patient E, sputum and pusisolates, which were identical, showed distinguishable PFGEpatterns. Table 2 shows the genetic relationship of MRSAisolated from nonblood specimens to blood isolates of MRSA.In total, 37 of 39 (94.9%) nonblood isolates of MRSA were

* Corresponding author. Mailing address: Department of Labora-tory Medicine, Nagasaki University School of Medicine, Nagasaki 852-8501, Japan. Phone: 81-95-849-7418. Fax: 81-95-849-7257. E-mail:hirakata@net.nagasaki-u.ac.jp.

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genetically related to blood isolates; these consisted of 33 in-distinguishable and four closely related isolates.

For the patients with MSSA bacteremia, all nonblood iso-lates, including the strains isolated from sputum (one strain), athroat swab (two strains), a nasal swab (one strain), pus (twostrains), ascites fluid (one strain), and the tip of a centralvenous catheter (two strains), were genetically indistinguish-able from blood isolates. For the patients with CoNS bactere-mia, 14 of 16 (87.5%) nonblood isolates, including the strainsisolated from sputum (2 strains), a throat swab (6 strains), anasal swab (2 strains), ascites fluid (1 strain), the tip of anintravenous catheter (1 strain), and feces (2 strains), weregenetically related to blood isolates, while 2 strains, 1 fromthroat swab and 1 from the tip of an intravenous catheter, weredifferent from the blood isolates from the respective patients.The 14 genetically related isolates consisted of 10 indistin-guishable, 2 closely related (isolates from a throat swab), and2 possibly related (isolates from a throat swab and ascites fluid)strains. An isolate of S. pneumoniae from cerebrospinal fluid

was genetically identical to a blood isolate from the samepatient. A strain of Enterococcus faecium isolated from sputumwas identical to the blood isolate from the same patient, whilea strain of Enterococcus faecalis isolated from the urine ofanother patient was different from a blood isolate from thatpatient.

Figure 2 shows PFGE patterns of P. aeruginosa strains fromsome of the patients with bacteremic episodes. In contrast tothose of MRSA, PFGE profiles of nonblood isolates of P.aeruginosa were frequently different from those of blood iso-lates. Although the genotypes of nonblood isolates were iden-tical to those of blood isolates for patients J through M, ge-netically distinct nonblood P. aeruginosa strains were isolatedfrom other patients. Although the genotypes of nasal andthroat isolates were the same for patient F, they differed fromthat of the blood isolate. A fecal isolate was genetically iden-tical to a blood isolate from patient G, while a different P.aeruginosa organism was isolated from the patient’s sputum.Although an isolate from pus was identical to a blood isolatefor patient H, sputum and throat isolates, showing the samegenotype, were distinguishable. Cross transmission betweenpatients K and L was suggested since the PFGE profiles of allisolates were genetically identical between the two. Table 3

FIG. 1. PFGE profiles of MRSA isolated from blood and nonblood specimens. Genomic DNA of MRSA strains was digested with restriction enzyme SmaI. Sampleswere ordered by set of blood isolate and nonblood isolate(s) from each patient. Lanes 1, 6, 10, 13, and 15 are blood isolates from various patients. Lanes 2 to 5 areisolates from sputum, the tip of a central venous catheter, pus, and feces from patient A, respectively. Lanes 7 to 9 are isolates from sputum and nasal and throat swabsfrom patient B, respectively. Lanes 11 and 12 are isolates from the tip of a central venous catheter and sputum from patient C, respectively. Lane 14 is an isolate frompus from patient D. Lanes 16 to 18 are isolates from pus, sputum, and a throat swab from patient E, respectively. M, molecular size marker.

TABLE 1. Number of bacteremic patients and related isolates usedfor the PFGE study

Organism(s)No. of

bacteremicpatients

No. ofrelatedisolatestesteda

MSSA 4 13MRSA 18 57CoNS 11 27S. pneumoniae 1 2Enterococci 2 4P. aeruginosa 19 45

Total 55 148

a Both blood and nonblood isolates from patients with bacteremia were in-cluded.

TABLE 2. Genetic relationships of MRSA strains isolated fromnonblood specimens and blood

PFGEinterpretation

No. of nonblood isolates from:

Sputum Throatswab

Nasalswab Pus Urine Feces Tip of

catheter Other

Indistinguishable 9 4 3 5 2 2 5 3Closely related 2 0 0 0 0 1 1 0Possibly related 0 0 0 0 0 0 0 0Different 0 0 1 0 0 0 1 0

Total 11 4 4 5 2 3 7 3

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shows the results of genetic analysis of P. aeruginosa isolatedfrom nonblood specimens and of blood isolates. The PFGEprofiles of two isolates from the tip of a central venous catheterdiffered from those of blood isolates. In total, 16 of 27 (59.3%)nonblood isolates of P. aeruginosa were genetically related toblood isolates, including 15 indistinguishable and 1 closely re-lated isolates. Additionally, more than two strains with differ-ent morphology or hemolysis activity were isolated from somesamples (one blood, one throat swab, and two sputa); theseshowed identical PFGE patterns. Genetically different non-blood isolates were rare for patients with bacteremia caused bygram-positive cocci (5 of 36 patients; 13.9%), while geneticallydistinct nonblood strains were isolated from 8 of 19 patients(42.1%) with P. aeruginosa bacteremia.

Most nonblood isolates of gram-positive cocci, especiallystaphylococci, were genetically related to blood isolates foreach patient. Therefore, antibiograms, the presence of resis-tance genes or specific virulence factors (e.g., toxic shock syn-drome toxin 1 or enterotoxins), and other information con-cerning nonblood isolates may help in the treatment ofpatients who have contracted bacteremia or other infections.

In contrast, nonblood isolates of P. aeruginosa frequentlydiffered in genotype from blood isolates. Moreover, three dif-ferent genotypes of P. aeruginosa were isolated from somepatients. Since P. aeruginosa widely exists both in the hospital

environment and in nature (e.g., in water, soil, and vegetables),it is possible that patients and healthy individuals are colonizedby several genetically different P. aeruginosa strains.

Of interest, for one patient with CoNS bacteremia and fortwo patients with P. aeruginosa bacteremia, the PFGE profilesof isolates from the tips of central venous catheters were dif-ferent from those of blood isolates. These findings suggest thattwo or more genetically distinct members of the same speciesof bacteria can invade the bloodstream of a patient.

Although surveillance cultures are performed prior to che-motherapy or organ transplantation in compromised hosts (3,8, 15), our results suggest that a single nonblood isolate of P.aeruginosa is not always informative. The findings of this studysuggest that several specimens from different sites should becultured and that antimicrobial susceptibility testing of theisolates should be performed.

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FIG. 2. PFGE profiles of P. aeruginosa isolated from blood and nonblood specimens. Genomic DNA of P. aeruginosa strains was digested with restriction enzymeSpeI. Samples were ordered by set of blood isolate and nonblood isolate(s) from each patient. Lanes 1, 4, 7, 11, 13, 15, 17, 19, and 21 are blood isolates from variouspatients. Lanes 2 and 3 are isolates from nasal and throat swabs from patient F, respectively. Lanes 5 and 6 are isolates from feces and sputum from patient G,respectively. Lanes 8 to 10 are isolates from sputum, a throat swab, and pus from patient H, respectively. Lanes 12, 14, 16, 18, 20, and 22 are isolates from the tip ofa central venous catheter from patient I, a throat swab from patient J, sputum from patient K, pleural effusion from patient L, urine from patient M, and feces frompatient N, respectively. M, molecular size marker.

TABLE 3. Genetic relationships of P. aeruginosa strains isolatedfrom nonblood specimens and blood

PFGEinterpretation

No. of nonblood isolates from:

Sputum Throatswab

Nasalswab Pus Urine Feces Tip of

catheter Other

Indistinguishable 3 4 1 3 2 1 0 1Closely related 0 0 0 1 0 0 0 0Possibly related 0 0 0 0 0 0 0 0Different 3 3 1 0 0 2 2 0

Total 6 7 2 4 2 3 2 1

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