Emerging Infectious Diseases
[Volume 5 No.3 / May - June 1999]


Dispatches

Emergence of Related Nontoxigenic Corynebacterium diphtheriae Biotype mitis
Strains in Western Europe

Guido Funke,* Martin Altwegg,* Lars Frommelt, and Alexander von
Graevenitz*
*University of Zurich, Zurich, Switzerland; and Endo-Klinik, Hamburg,
Germany

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      We report on 17 isolates of Corynebacterium diphtheriae biotype
      mitis with related ribotypes from Switzerland, Germany, and
      France. Isolates came from skin and subcutaneous infections of
      injecting drug users, homeless persons, prisoners, and elderly
      orthopedic patients with joint prostheses or primary joint
      infections. Such isolates had only been observed in
      Switzerland.

Nontoxigenic Corynebacterium diphtheriae strains were recovered from
approximately 1 per 1,000 throat swabs from immunized British military
personnel in Germany from 1993 to 1995 (1). Such nontoxigenic C.
diphtheriae biotype mitis isolates had been described in skin, throat, and
blood cultures of Swiss injecting drug users; 32 of the isolates belonged
to the same clone (2,3). Our study demonstrates that this clone and closely
related clones occurred between 1990 and 1997 in two other European
countries, and only sometimes in persons with poor hygiene.

Five C. diphtheriae isolates came from two laboratories in Zurich and Bern,
Switzerland; 11 from four laboratories in Hamburg, Germany; and 1 from
Paris, France (Table). They were identified in Zurich as C. diphtheriae
biotype mitis (4); that is, they were nonlipophilic, were nitrate
reductase-positive, and did not ferment glycogen. By polymerase chain
reaction techniques (5), the diphtheria toxin gene was not detected in any
isolate. For some isolates, the Elek test was also performed; it was
consistently negative. For ribotyping, DNA was isolated, digested with
either EcoRI or PvuII, electrophoresed, blotted, and probed for rDNA as
described elsewhere (2,3). Disk susceptibility testing to tetracycline and
MIC determinations were done according to National Committee for Clinical
Laboratory Standards methods (6). All other bacteria isolated were also
identified and serotyped in Zurich.

Table. Nontoxigenic Corynebacterium diphtheriae isolates

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                               Ribotyping
                               pattern(sup c)  
                               ----------- Patients    Clinical
Isolate  Date       Place of               sex, age   diagnosis/     Other bacteria     Underlying
no.    isolated    isolation   EcoRI PvuII   (yr)       source          isolated         conditions
		(sup a)   (sup b)  
-----------------------------------------------------------------------------------------------------
2012    1990       Paris         A     C     m, 6   Endocarditis/                    Skin lesions,
                                                    blood culture                     scabies

2410    2/1995     Hamburg       A     B    m, 30   Ulcus/lower    Staphylococcus     Prisoner
                                                    leg            aureus,
                                                                   Streptococcus
                                                                     pyogenes
2689    3/1995     Hamburg       A     A    f, 82   Puncture/hip   S. epidermidis,    Total hip joint
                                                    joint          Corynebacterium    endoprosthesis
                                                                   pseudodiphtherit-
                                                                   icum

1682    9/1995     Hamburg       A     B    m, 45   Wound swab     S. aureus,         Homelessness
                                                                   S. pyogenes

1935    3/1996     Hamburg       A     B    m, 58   Wound swab     S. aureus,         Alcoholism,
                                                                   S. pyogenes        homelessness

1836    3/1996     Hamburg       A     B    m, 45   Abscess/lower  S. aureus,         Injecting
                                                    leg            S. pyogenes        drug use

2661    3/1996     Hamburg       A     A    m, 35   Arthroscopy    Peptostreptococcus Osteosynthesis,
                                                    aspirate/knee
                                                    joint          prevotii,P. magnus arthrotomy,
                                                                                      knee joint
                                                                                      after trauma

2658    4/1996     Hamburg       A     A    f, 76   Fistula/hip    S. aureus          Total hip
                                                    joint                             joint
                                                                                      endoprosthesis
2674    5/1996     Hamburg       A     A    f, 62   Aspirate/hip   Streptococcus      Total hip joint
                                                    joint          group C
                                                                                      endoprosthesis
2670    7/1996     Hamburg       A     A    f, 81   Aspirate/knee                    Total knee
                                                    joint                             joint
                                                                                      endoprosthesis
2667    7/1996     Hamburg       A     A    m, 23   Swab/hip joint Coagulase-negative Total hip joint

                                                                   staphylococci      endoprosthesis
2413    11/1996    Hamburg       A     B    m, 36   Ulcus/lower    S. aureus,         Prisoner
                                                    leg            Streptococcus
                                                                   group C/G
2446    12/1996    Zurich        A     A    f, 38   Wound/upper    S. aureus, S.      Injecting drug
                                                    leg            pyogenes           use

2464    1/1997     Zurich        A     A    m, 39   Ulcera/lower   Escherichia coli,  Injecting drug
                                                    arm                               use
                                                                   S. aureus, S.
                                                                   pyogenes
2473    1/1997     Zurich        A     A    m, 38   Ulcera/leg     E. coli,           Injecting drug
                                                                   Streptococcus      use
                                                                   group C, G
2475    1/1997     Zurich        A     A    m, 31   Ulcus/pretibialPseudomonas        Injecting drug
                                                                   aeruginosa,  E.    use
                                                                   coli,
                                                                   Streptococcus
                                                                   group C, G

480     5/1997     Bern          A     A    m, 39   Ulcus/upper legS. aureus,         Injecting
                                                                   Clostridium        drug use
                                                                   perfringens,
                                                                   mixed
                                                                   anaerobic flora
-----------------------------------------------------------------------------------------------------
(sup a)Month/Year
(sup b)Paris, France; Hamburg, Germany; Zurich, Switzerland; Bern, Switzerland.
(sup c)DNA was isolated, digested with either EcoRI or PvuII, electrophoresed, blotted, and probed for
DNA. Three distinct ribopatterns emerged, each with eight bands: patterns A, B, and C.

Many (10 [59%] of 17) C. diphtheriae isolates were from skin or
subcutaneous infections (wounds, ulcers) in Swiss patients and were found
with Staphylococcus aureus, Streptococcus pyogenes, group C/G streptococci,
or (sometimes) with gram-negative rods. Most patients in this subgroup were
injecting drug users, homeless persons, prisoners, and (with one exception)
men with a mean age of 40 (30 to 58) years. Another subgroup (from Germany)
consisted of six patients with joint or bone infections (mentioned briefly
in an earlier publication on coryneform bacteria from such infections; a
seventh patient had C. diphtheriae biotype gravis [7]). The six had been
hospitalized at the Endo Clinic in Hamburg, which specializes in orthopedic
surgery. Four had had implantations of hip endoprostheses and had been
admitted for prosthetic infections (with coagulase-negative staphylococci
or S. aureus); one pure culture of C. diphtheriae biotype mitis was
obtained from a patient with a knee prosthesis, and one mixed culture with
Peptostreptococcus magnus and Peptostreptococcus prevotii was obtained from
a patient who had a purulent knee infection after a fracture. Their average
age was 60 (23 to 82) years and, to our knowledge, none was a drug user.
While the Hamburg isolates were recovered only once from every patient,
their isolation dates stretched from March 1, 1995, until July 26, 1996.
Three were isolated on the day of admission, and all of them were isolated
by different technologists. Finally, one child (from France) had
endocarditis with C. diphtheriae biotype mitis.

All isolates had identical antimicrobial susceptibility patterns. They were
susceptible to amoxicillin (MIC, 0.25 g/ml), amoxicillin-clavulanic acid
(0.06 g/ml), chloramphenicol (2 g/ml), ciprofloxacin (0.25 g/ml),
clarithromycin (0.03 g/ml), clindamycin (0.25 g/ml), imipenem (0.03
g/ml), penicillin (0.25 g/ml), and vancomycin (1 g/ml). In contrast, all
strains were resistant to tetracycline in the disk diffusion test
(inhibition zone diameter 10 mm to 11 mm); their MICs for tetracycline,
doxycycline, and minocycline were 64 g/ml, 16 g/ml, and 16 g/ml,
respectively. While this type of isolated tetracycline resistance was
typical for the isolates from Swiss injecting drug users (3), tetracycline
resistance in nontoxigenic C. diphtheriae has otherwise very rarely been
observed in Europe (8). It has, however, been reported in toxigenic C.
diphtheriae isolates from Indonesia and, rarely, from Canada (9). The
mechanism conferring this resistance in our strains has not been
investigated.

On analysis with restriction enzyme Pvu II, three different ribopatterns
with eight bands each were found among the strains (Figure). These
patterns, though distinct, shared five (A vs. C, B vs. C) and six (B vs. A)
bands, respectively, and, therefore, may be considered related, as they
would be if criteria for pulsed-field gel electrophoresis were applied
(10). This view is supported by the fact that all strains had identical
EcoRI patterns (not shown). All Swiss isolates were identical with the use
of both enzymes, whereas both the Swiss pattern and a second pattern were
found among the Hamburg isolates. The third pattern was found exclusively
in the single French strain. The staphylococcal and streptococcal strains
were not typed; they are no longer available to us.

Our isolates thus   
resemble those      Figure. Ribosomal RNA gene restriction patterns
reported from       obtained by using restriction enzyme Pvu II of
Switzerland between Corynebacterium diphtheriae isolates belonging to
1990 and early 1996 ribotypes B (lanes 1 to 3), A (lanes 4 to 6 and 8),
(2,3), which were   and C (lane 7).
also often
accompanied by S.
aureus or beta-hemolytic streptococci. Such nontoxigenic C. diphtheriae
mitis may cause endocarditis, arthritis, and osteomyelitis (11,12). Most of
the 52 isolates from France (11), examined with restriction enzymes
different from ours and not available to us, also belonged to one ribotype.
Their relatedness to our strains is unknown; however, they were largely
tetracycline-susceptible. The two throat isolates from St. Petersburg,
Russia, associated with a fatal diphtherialike disease (12) were not typed
or tested for antibiotic susceptibility.

The origin of the isolates we describe is unknown. They may have been
present (but unrecognized) in the population for a long time. The mode of
transmission is most likely common use of drug paraphernalia in the
injecting drug use cases and in the endocarditis case; transmission is very
difficult to explain in the orthopedic infection subgroup. Although the
Swiss and the Danish borders are not close, migration and contacts are not
uncommon among injecting drug users. The isolates may also have been
distributed through the drugs themselves, as recently reported for S.
pyogenes in Switzerland (13).

Our study may be representative for Switzerland and Germany, but since
submitting C. diphtheriae strains to a central reference laboratory in
these countries is not mandatory, we cannot estimate the frequency of these
strains. These strains may also have spread to other European countries;
this hypothesis can only be tested in a large multicenter study of European
diphtheria reference laboratories.

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Acknowledgments

      We thank J. Lthy-Hottenstein and V. Pnter-Streit for excellent
technical assistance and P. Das for her help in securing the strains from
Hamburg. G. Funke is recipient of a European Society for Clinical
Microbiology and Infectious Diseases research fellowship.

      Dr. Funke is director of clinical microbiology at Grtner &
Colleagues Laboratories in Weingarten, Germany. His areas of expertise are
clinical and systematic bacteriology. Research interests include taxonomy
and disease associations of coryneform bacteria and actinomycetes, as well
as rapid methods for identification and susceptibility testing of medically
relevant bacteria.

      Address for correspondence: Alexander von Graevenitz, Department of
Medical Microbiology, University of Zurich, Gloriastra sse 32, CH-8028
Zurich, Switzerland; fax: 411-634-4906; e-mail: avg@immv.unizh.ch.

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Emerging Infectious Diseases
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