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Keywords:

  • Extremely low-birth-weight infant;
  • methicillin-resistant Staphylococcus aureus;
  • necrotizing fasciitis;
  • Panton–Valentine leukocidin;
  • staphylococcal enterotoxin

Abstract

  1. Top of page
  2. Abstract
  3. Acknowledgement
  4. Transparency Declaration
  5. References

Clin Microbiol Infect 2010; 16: 289–292

Abstract

Necrotizing fasciitis due to methicillin-resistant Staphylococcus aureus (MRSA) is an uncommon but life-threatening infection, and has mainly been reported as occurring in adults and the elderly. Recently, infant cases involving Panton–Valentine leukocidin (PVL)-positive community-acquired MRSA have been noted. Here, a case of fatal necrotizing fasciitis with sepsis and disseminated intravascular coagulation in an extremely low-birth-weight infant is described. The causative agent was the hospital-acquired MRSA New York/Japan clone carrying the spa variant gene and nine staphylococcal enterotoxin (SE) genes. These data suggest that a high-level combination of SEs and other virulence factors, but not PVL, could contribute to the pathogenesis of fatal necrotizing fasciitis.

Necrotizing fasciitis is an uncommon but life-threatening infection, in which progressive necrosis occurs in superficial fascia and adjacent subcutaneous tissue [1]. Mortality rates reach 6–76% [1,2]. It is often polymicrobial (including Staphylococcus aureus) but not always [3].

Methicillin-resistant Saureus (MRSA) has been a major cause of nosocomial infections since 1961 [4] and community-acquired MRSA has recently emerged [5], causing skin and soft-tissue infections [5], and often producing Panton–Valentine leukocidin (PVL) [6]. Here, a fatal case of MRSA-caused necrotizing fasciitis in an extremely low-birth-weight (ELBW) infant is described, as well as the molecular characteristics of the MRSA isolate.

An ELBW (652 g) infant born at 24 weeks and 2 days of gestation was transferred to the neonatal intensive care unit of a hospital just after delivery, and placed under artificial respiratory management. The blood pressure and urine volume decreased from days 0 to 7 after birth. Vasopressin administration was initiated on day 6 after birth, in addition to hydrocortisol. Hyperkalaemia persisted, with oliguria, for which glucose insulin (GI) treatment was continued until day 6. Intracranial haemorrhage was detected by transcranial ultrasound examination on day 2, and resolved by blood transfusion on day 5. Although the patent ductus arteriosus became symptomatic on day 4, indomethacin treatment was postponed until haemostasis of the intracranial haemorrhage on day 8, and the duct was closed on day 10. Ampicillin venous injection was initiated when C-reactive protein (CRP) levels rose slightly (0.15 mg/dL) on day 10. CRP levels further rose to 2.5 mg/dL on day 11, and sepsis was suspected. Amikacin was additionally administered, but leucocyte and platelet counts decreased (5500/mL and 1.6 × l04/mL, respectively) on day 12, suggesting complication by disseminated intravascular coagulation (DIC), for which granulocyte-colony stimulating factor (G-CSF) and gabexate mesylate administration was initiated. Discoloration of the patient’s right leg occurred within 12 h. The discoloration (purple) of the skin was first noticed below the ankle around the area of percutaneous central venous catheter insertion for venous infusion (day 11), and rapidly spread to the proximal part of the right extremity. Careful examination of the right leg after removal of the catheter revealed that the point of catheter insertion was black due to necrosis of the skin. Hyperkalaemia occurred again on day 12, and GI treatment was reinstated. On day 12, MRSA was detected upon culture of blood and the right leg necrotic region, and the antimicrobial treatment was switched to panipenem and vancomycin. Wide QRS and ventricular arrhythmia were observed upon electrocardiography on day 13. The blood pressure fell and bradycardia appeared, and the patient died in the evening of the same day.

Molecular typing of MRSA was performed as described previously [7]. Multi-locus sequence typing was conducted using seven housekeeping genes. The spa type was analysed by sequencing of the PCR product of the spa gene [encoding for Spa (protein A)]. Detection of the accessory gene regulator (agr) allele group was according to PCR. The staphylococcal cassette chromosome mec (SCCmec) types (I–V) were analysed by PCR. Coagulase typing was conducted using a staphylococcal coagulase antiserum kit (Denka Seiken, Tokyo, Japan). Analysis of the virulence genes (three leukocidin genes including the PVL gene, five haemolysin genes, 17 staphylococcal enterotoxin (SE) genes, one putative SE gene, three exfoliative toxin genes, and 14 adhesin genes) was according to PCR. Susceptibility testing of bacterial strains was carried out using the agar dilution method according to previous procedures [7]. Antimicrobial agents included oxacillin, gentamicin, kanamycin, arbekacin, vancomycin, teicoplanin, linezolid, tetracycline, minocyclin, erythromycin, clindamycin, levofloxacin, fosfomycin, trimethoprim, sulfamthoxazole, fusidic acid, rifampicin, and mupirocin.

The molecular characteristics of the isolated MRSA (strain NN33) are summarized in Table 1. It belonged to ST5 and exhibited spa387, agr2, SCCmecII, and coagulase type II, indicating a spa variant (spa387) of the New York/Japan clone [7–9,12]. The sequence of the spa gene of strain NN33 (GenBank accession no. AB467285) demonstrated a shorter stem region (short sequence repeat) than the stem region of spa2 MRSA strains. Regarding other virulence genes, strain NN33 was negative for the PVL gene, but positive for the enterotoxin gene cluster (egc) carrying seg, sei, sem, sen, and seo, as the New York/Japan clone [7,12]. Moreover, it carried the superantigen-encoding pathogenicity island (SaPIm1/n1), possessing the genes tst [which codes for toxic shock syndrome toxin 1 (TSST-1)], sec and sel, as the Japanese type of the New York/Japan clone [7,9]. Strain NN33 was also positive for the sep gene, and was multiple-drug resistant (Table 1).

Table 1.   Characteristics of MRSA strain NN33 and comparison with the Japanese and US type strains of the New York/Japan clone
Type, virulence gene, or drug resistanceJapanese typeUS type
Strain NN33Strain Mu50Strain BK2464a
  1. G, gentamicin; K, kanamycin; V, vancomycin; T, tetracycline; M, minocycline; E, erythromycin; C, clindamycin; L, levofloxacin; F, fosfomycin; Tr, trimethoprim; R, rifampicin.

  2. aStrain BK2464 was kindly provided by H. de Lencastre [8].

  3. bspa-types from the Ridom Spa Server are shown in parentheses.

  4. cThe repeat succession for spa387: T1M1G1M1K1.

  5. dThe repeat succession for spa2: T1J1M1B1M1D1M1G1M1K1.

  6. eSplit hlb gene due to insertion of a phage [9].

  7. fWhen 11 strains, including strains Mu50 (from a surgical wound infection) and N315 (from a pharyngeal infection), whose genome sequences were determined [9], two isolates from toxic shock syndrome (TSS), and seven isolates from neonatal TSS-like exanthematous disease (NTED) [10], were examined, one of 11 was positive for sep, and three of 11 were positive for sea, indicating that positive cases are rare.

  8. gThe data of Japanese type strain N315 were the same as those of Mu50, except the data shown in parentheses.

  9. hc12ag, core 12 adhesin genes shared by all strains: icaA, icaD (for biofilm formation); eno (for laminin-adhesin); fnbA, fnbB (for fibronectin-adhesin); ebpS (for elastinadhesin);

  10. clfA, clfB, fib, sdrC, sdrD, sdrE (for fibrinogen) [11].

  11. iUnderlining indicates intermediate resistance.

Type
ST555
spab387c (t653)2d (t002)2d (t002)
agr222
SCCmec typeIIIIII
Coagulase typeIIIIII
Virulence gene
 Leukocidin
  lukE-lukD+++
  lukPVSF, lukM
 Haemolysin
  hla, hlg, hlg-v+++
  hld+++
  hlb(−)e(−)e(−)e
 Enterotoxin
  SaPIm1/n1 (tst, sec, sel)++
  egc (seg, sei, sem, sen, seo)+++
  sep+f(+)f,g+
  sea+f(−)f,g
  sed, sej
  seb, see, seh, sek, seq, seu
 Exfoliative toxin
  eta, etb, etd
 Adhesin
  c12agh+++
  cna, bbp
Drug resistanceK, T, Mi, E, C, L, FG, K, Vi, T, Mi, E, C, L, F, R (K, E, C)gE, C, L, Tr

Although patients with necrotizing fasciitis are mostly adults or the elderly [1,13], neonatal MRSA necrotizing fasciitis has been noted recently in Taiwan [14] and the USA [15,16]. In the latter cases, the causative agents were community-acquired MRSA (USA300 clone) with the PVL gene.

To the best of our knowledge, this study demonstrates the first fatal case of necrotizing fasciitis with sepsis and DIC in an ELBW infant. The causative agent was the pandemic New York/Japan clone (PVL-negative hospital-acquired MRSA), indicating that PVL is not essential for the pathogenesis of necrotizing fasciitis. SEs are superantigens [17]. The isolated MRSA (strain NN33) carried genes for a high-level combination of SEs (nine SEs coded for by SaPIm1/n1 [18], egc [19], and sep). Among those, TSST-1 may suppress the mobility of polymorphonuclear neutrophils to infection sites [18], allowing MRSA to invade tissues. Moreover, since Spa protein binds to the tumour necrosis factor-α receptor of tissue cells [20], Spa protein, with a shorter stem, of strain NN33 (spa387) may allow MRSA to get closer to and damage tissue cells effectively.

In addition to the prematurity (i.e. ELBW) of the patient as a risk factor, a high-level combination of SEs (including TSST-1), together with adhesins and Spa (spa387 variant), could have contributed to the pathogenesis of fatal necrotizing fasciitis with sepsis and DIC.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Acknowledgement
  4. Transparency Declaration
  5. References

We thank H. de Lencastre for the BK2464 strain.

Transparency Declaration

  1. Top of page
  2. Abstract
  3. Acknowledgement
  4. Transparency Declaration
  5. References

This study was supported in part by a grant from Niigata University. There are no conflicting interests.

References

  1. Top of page
  2. Abstract
  3. Acknowledgement
  4. Transparency Declaration
  5. References
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