Emerging Campylobacter spp.: The tip of the iceberg

Clinical Microbiology Newsletter Vol. 28, No. 7 April 1, 2006 Emerging Campylobacter spp.: the Tip of the Iceberg Albert J. Lastovica, Ph.D., Clinical Laboratory Science (Medical Microbiology) and IIDMM, University of Cape Town, Cape Town, South Africa Abstract Campylobacter jejuni is universally recognized as the most common bacterial cause of human gastroenteritis. This organism is also associated with septicemia, meningitis, and the post-infective sequelae of the Guillain-Barré syndrome and reactive arthritis. Interest is increasing in Campylobacter species other than C. jejuni and their roles in human and animal disease. However, these emerging campylobacteria are dramatically under-isolated because of the application of less than optimal laboratory protocols. A Cape Town protocol in which stool is filtered onto antibiotic-free culture media and incubated in a hydrogen-enriched, microaerobic atmosphere is a simple and cost-effective means of optimizing the recovery of all species of Campylobacter, as well as species of the related genera Arcobacter and Helicobacter, from stool, blood, and other clinical samples. Subsequent biochemical identification by means of a flowchart easily identifies presumptive Campylobacter isolates to the species level. As emerging Campylobacter spp. are dramatically under-isolated, the true disease potential of these organisms is unknown at present. Introduction “Only one percent of human illness due to Campylobacter is caused by Campylobacter species other than C. jejuni or C. coli.” (http://www.cdc.gov/ campylobacter) Campylobacter spp. are universally acknowledged as the most common bacterial cause of enteritis worldwide. Of the 17 recognized and named species of the genus Campylobacter, 14 species — Campylobacter coli, Campylobacter concisus, Campylobacter curvus, Campylobacter fetus, Campylobacter hominis, Campylobacter hyointestinalis, Campylobacter jejuni, Campylobacter lanienae, Campylobacter lari, Campylobacter mucosalis, Campylobacter rectus, Campylobacter showae, Campylobacter sputorum, and Campylobacter upsaliensis — have been isolated from symptomatic and asymptomatic Mailing address: Albert J Lastovica, Ph.D., Medical Microbiology, IIDMM,University of Cape Town, Medical School, Anzio Road, Observatory, 7925, Cape Town, South Africa. Tel.: (+27) 21 406 6083. Fax: (+27) 21 448 8150. E-mail: lastoaj@mweb.co.za Clinical Microbiology Newsletter 28:7,2006 humans. C. jejuni is by far the most commonly isolated Campylobacter species. C. jejuni has two subspecies, C. jejuni subsp. jejuni and C. jejuni subsp. doylei. Although infrequently isolated, C. jejuni subsp. doylei has been associated with gastroenteritis and septicemia in pediatric patients (1). C. jejuni subsp. jejuni is overwhelmingly the most frequently isolated Campylobacter worldwide and is associated with enteritis, particularly in young children, in both developed and developing countries. This bacterium is recognized as the most common antecedent pathogen associated with the Guillain-Barré syndrome and reactive arthritis. C. jejuni subsp. jejuni has also been associated with other clinical conditions, such as septicemia, meningitis, the hemolyticuremic syndrome, pancreatitis, and abortion. The role of C. jejuni subsp. jejuni in human disease is well established, but the clinical relevance of other Campylobacter species (emerging campylobacteria) is far less well understood because relatively few strains of these organisms have been isolated from clinical mater© 2006 Elsevier ial. The paucity of isolated Campylobacter strains is undoubtedly due to a variety of reasons (Table 1). Current isolation protocols for Campylobacter spp.are biased in favor of the isolation of C. jejuni subsp. jejuni and C. coli. Less than optimal conditions of temperature, atmosphere, and incubation time contribute to the failure to isolate emerging Campylobacter spp. Most diagnostic microbiology laboratories use commercially available isolation plates containing antibiotics for suppressing the growth of bacteria other than Campylobacter spp. However, these selective media contain components that inhibit the growth of emerging Campylobacter spp., as has been extensively documented by Loades et al. (2); varying susceptibilities by agar disk diffusion are shown in Fig. 1A. The isolation of C. upsaliensis and other 0196-4399/00 (see frontmatter) 49 emerging campylobacteria from stool and other clinical material is unsuccessful when media containing antibiotics to which these organisms are susceptible are used. Some emerging Campylobacter spp., such as C. concisus, C. showae, C. rec- tus, and C. curvus, have an essential growth requirement for hydrogen or formate (Fig. 1B). They are found in the gingival flora of the human mouth, notably in periodontal pockets of diseased gums, although a causal role for these organisms has not been established. Table 1. Reasons for failure to detect emerging campylobacteria and related organisms • Preconception that emerging Campylobacter spp. are not commonly found • Lack of appreciation of the growth and biochemical characteristics of emerging Campylobacter spp. • Use of antibiotic-containing selective media that may inhibit growth of emerging Campylobacter spp. • Inappropriate isolation temperature • Inappropriate isolation atmosphere • Inappropriate length of time for isolation A B C D Figure 1. (A) A C. upsaliensis isolate showing a 4-cm inhibitory zone for nalidixic acid (30-µg disk) on the left and a 5-cm inhibitory zone for cephalothin (30-µg disk) on the right. (B) Clinical isolates of C. concisus and C. jejuni subsp. jejuni grown on tryptose blood agar under microaerophilic conditions (right) and H2-enriched microaerophilic conditions (left). (C) Variation of colony morphology for clinical isolates of C. jejuni subsp. jejuni (Cjj), C. concisus (C con) and C. hyointestinalis (C hyo). (D) Stool filtration onto antibiotic-free tryptose blood agar: stool application (left) and the isolation plate (right) after 48 h of incubation at 37°C in an H2-enriched microaerophilic atmosphere. 50 0196-4399/00 (see frontmatter) © 2006 Elsevier These Campylobacter species have been isolated from the stools of patients suffering from gastroenteritis, septicemia, and other clinical conditions (3). Hydrogen-dependent organisms are extremely difficult, if not impossible, to recover by the standard culture techniques currently employed in most routine diagnostic laboratories. Some diagnostic laboratories use 42°C as a primary incubation temperature, which allows growth of C. jejuni subsp. jejuni and C. coli but not of other species, such as C. fetus or C. hyointestinalis, which grow at 37°C but not 42°C. C. jejuni subsp. jejuni and C. coli contaminate chicken which has an internal temperature of 42°C. This is probably the historical precedent for using 42°C as a primary incubation temperature. For clinical specimens, it is recommended that incubation at 37°C is a more appropriate temperature, as all Campylobacter spp. infecting humans can be isolated and maintained at this temperature. Up to 17% of clinical samples may have two to five distinct species of Campylobacter or species of the related genera Arcobacter and Helicobacter identified on the primary isolation plate (4). C. jejuni subsp. jejuni may be coisolated with an emerging Campylobacter sp. Different species of Campylobacter and related genera can often be detected initially by differences in colony morphology (Fig. 1C) and subsequently confirmed by biochemical testing (Fig. 2). Most strains of C. jejuni subsp. jejuni and C. coli are relatively fast growing, while other Campylobacter species, such as C. upsaliensis, C. rectus, C. hyointestinalis, and C. concisus, are much slower growing. A bench technologist might isolate a C. jejuni subsp. jejuni strain after 2 days and promptly discard the isolation plate, which, if kept and re-incubated for an additional 2 to 4 days, could subsequently yield Clinical Microbiology Newsletter 28:7,2006 Figure 2. Biochemical flowchart for the identification of Campylobacter, Helicobacter, and Arcobacter species from clinical material. This procedure is used after the presumptive isolate is determined to be gram negative and negative for the L-ALA test, and also if the isolate can grow under aerobic conditions or if it has an essential requirement for a microaerophilic or an H2-enriched microaerophilic atmosphere. The biochemical tests outlined are standard diagnostic tests for Campylobacter spp. Useful additional tests are nitrate reductase, catalase, lead acetate, and pyrazinamidase. Nalidixic acid (30 µg disk) and cephalothin (30-µg disk) have limited diagnostic use, as an increasing number of Campylobacter strains are resistant or intermediate for these antibiotics. The complete biochemical profiles of C. jejuni subsp. jejuni, emerging Campylobacter spp., Arcobacter spp., and Helicobacter spp. are detailed in reference 1. additional strains of Campylobacter or related genera. Reservoirs of Emergent Campylobacters C. jejuni subsp. jejuni infection is usually acquired from contaminated food, particularly poultry, beef, sheep, and unpasteurized milk. Reservoirs of newly described emerging Campylobacter spp. have been found in production animals, such as pigs, sheep, cattle, and poultry. Emergent campylobacteria have also been isolated from pets and wild animals, such as dogs, cats, hamsters, foxes, monkeys, rodents, and seals. However, non-mammalian species, such as wild birds and shellfish, have recently been implicated as reservoirs for these organisms (3). Whether emerging Campylobacter spp., if present in the food chain, are a potential health hazard Clinical Microbiology Newsletter 28:7,2006 must still be determined. This question is currently being addressed. Information in this regard can be obtained on the website http://www.campycheck.org. Humans appear to be the only known reservoir of C. concisus and C. gracilis. Rivers, seawater, and groundwater are known to harbor Campylobacter and Arcobacter spp. (2). Moore et al. (5) have provided a brief but informative review of emerging Campylobacter spp., including the recently described C. hominis, C. lanienae, and C. sputorum bv. paraureolyticus, all of which have been isolated from human and animal feces. Little is known about virulence factors in emerging campylobacteria, as almost all studies on Campylobacter spp. have been done on C. jejuni subsp. jejuni. Pathogenic mechanisms associated with acute intestinal infection in campylobacteriosis are poorly understood but most probably involve adherence, invasion of intestinal cells, and toxin production. Sylvester et al. (6) demonstrated that C. upsaliensis is capable of binding to Chinese hamster ovary (CHO) cells in cell culture. These authors also detected 50- to 90-kDa surface proteins on C. upsaliensis isolates that were capable of binding to phosphatidylethanolamine, a putative cell membrane receptor. Fouts et al. (7) have discovered a novel putative licABCD virulence locus in C. upsaliensis with significant similarity to genes present in Streptococcus, Haemophilus, and Neisseria spp. Genes in these microorganisms encode proteins © 2006 Elsevier 0196-4399/00 (see frontmatter) Virulence Factors of Emergent Campylobacteria 51 involved in the acquisition of choline, the synthesis of phosphocholine (PCho), and finally, the transfer of PCho to the teichoic and lipoteichoic acids of lipooligopolysaccharide (a component of the outer membrane of some gramnegative bacteria that is similar to lipopolysaccharide in structure but with a shorter O antigen carbohydrate chain) to facilitate bacterial attachment to host cells. Cytolethal distending toxins from enteropathogenic Escherichia coli disrupt the barrier function of host intestinal epithelial tight junctions. Pickett et al. (8) confirmed the presence of a cytolethal distending toxin homologue in C. upsaliensis. However, the appropriate cdt genes have yet to be cloned. Efficient Isolation of Emergent Campylobacter spp. The Cape Town protocol for the isolation of Campylobacter spp. from stool (Table 2) uses filtration through a membrane filter onto an antibiotic-free blood agar plate (Fig. 1D) and subsequent incubation at 37°C in an H2-enriched microaerobic atmosphere. This protocol has dramatically increased both the number of strains and the number of Campylobacter spp. and species of the related genera Arcobacter and Helicobacter isolated from diarrheic stools of pediatric patients (3). By using antibiotic-containing selective media at the Red Cross Children’s Hospital (RXH) in Cape Town, South Africa, 7.1% of diarrheic stools from pediatric patients were positive for Campylobacter spp. After the implementation of the Cape Town protocol, the number of Campylobacter-positive stools rose to 21.8% (4). For the first time, C. upsaliensis, C. concisus, C. hyointestinalis, C. rectus, Helicobacter fennelliae, Helicobacter cinaedi, and Arcobacter butzleri were isolated from the diarrheic stools and blood cultures of pediatric patients. A modification of the Cape Town protocol (no filter is used) has proved to be efficient in isolating species of Campylobacter and related genera from blood, pleural aspirates, and other clinical material, as well as Helicobacter pylori from gastric biopsy specimens. The Cape Town protocol has been evaluated recently at the International Center for Diarrhoeal Diseases Research, B: Center for Health and Population 52 0196-4399/00 (see frontmatter) Table 2. Essential features of the Cape Town protocol for the isolation of Campylobacter and the related genera Arcobacter and Helicobacter from stoola 1. Stool: prepare a watery emulsion in sterile saline. Mucoid samples should be vortexed. 2. Aseptically place a 0.6-µm-pore-size membrane filter (Schleicher & Schuell ME 26) directly onto a triptose blood agar (TBA) plate (Oxoid CM233, 10% unlysed horse or sheep blood). 3. Flood the central area of filter with the suspension using a transfer pipette. 4. Reflood two or three times. 5. Remove and discard filter (±15 min). Incubate the plate as soon as possible under H2enriched microaerobic conditions at 37ºC.b 6. Incubate plate for 6 days, examining every 2 days. Do not discard or ignore the primary plate once growth has been obtained, as more than one species may be present which have different growth rates. Morphologically different colonies (size, color, time of appearance, etc.) may indicate mixed infections. If so, subculture to purify. 7. Subculture single colonies and use the L-ALA test as a prescreen (Table 3). 8. A single colony should be subcultured on TBA and aerobically cultured for 2 days to establish whether there is growth, which would indicate that it is an Arcobacter strain. 9. Follow the biochemical flowchart (Fig. 2) for identification. a This protocol may be modified to use blood or other clinical samples. Gastric biopsy material: gently spread the biopsy sample on the surface of a TBA plate using a swab moistened with sterile saline. Blood cultures: Apply ~0.2 ml of the mixture taken from the blood culture bottle over the surface of a TBA plate. DO NOT USE A FILTER. b H2–enriched microaerobic conditions are obtained by using anaerobic sachets (Oxoid BR38 or BBL 70304) with NO CATALYST. Research, in Dhaka, Bangladesh. As a result, the number of species and the number of Campylobacter strains (including C. jejuni subsp. jejuni) isolated in their laboratory has increased dramatically (9). As outlined in Table 2, the use of the Cape Town protocol is simple, as is the subsequent identification by means of the biochemical flowchart (Fig. 2). As species of Helicobacter and Arcobacter are often co-isolated with Campylobacter spp. from clinical specimens, and as they are morphologically similar to Campylobacter, it is necessary to differentiate these organisms from Campylobacter. The use of the biochemical flow chart in Fig. 2 readily does this. Campylobacter, Helicobacter, and Arcobacter strains can easily be differentiated from other gram-negative and gram-positive bacteria. L-Alanine aminopeptidase (L-ALA) is found in most gram-negative bacteria, but not in gram-positive bacteria or in Campylobacter, Helicobacter or Arcobacter species (10). The Oxoid Biochemical Identification System (O.B.I.S.; Oxoid, Ltd., Basingstoke, Hampshire, U.K.) uses L-alanyl-7-amino-4-methylcoumarin as a substrate, while the Fluka detection system (Fluka GmbH, Buchs, Switzerland) uses L-alanine4-nitroanilide. Test results from both © 2006 Elsevier detection systems were identical (10). For laboratories without Gram-staining ability, an alternative test is the potassium hydroxide test, which lyses the cell wall of gram-negative bacteria, releasing DNA. Gram-positive bacteria are not lysed in this test. Table 3 illustrates the results of the application of the L-alanine aminopeptidase test and the potassium hydroxide DNA “string” test for determining the Gram stain reactions of bacteria (10). These two simple tests provide an easy and rapid means of differentiating Campylobacter, Helicobacter, and Arcobacter from other gram-negative and gram-positive bacteria. While Campylobacter and Helicobacter strains require microaerophilic or H2-enriched microaerophilic growth conditions, strains of Arcobacter will grow under these conditions, as well as aerobically. Aerobic growth is a defining characteristic of the genus Arcobacter. Incubation under aerobic conditions will readily and simply differentiate Arcobacter from Campylobacter and Helicobacter strains. These preliminary tests are useful before starting biochemical species identification of presumptive Campylobacter strains (Fig. 2). Clinical Associations In contrast to the statement on the CDC website quoted in the introduction, Clinical Microbiology Newsletter 28:7,2006 C. jejuni subsp. jejuni has not been the major Campylobacter species isolated from pediatric patients with enteritis and septicemia in Cape Town over the last 15 years (Table 4). Two-thirds of the Campylobacter isolates from the diarrheic stools of pediatric patients at RXH were species other than C. jejuni subsp. jejuni. When directly compared to those of the recognized pathogen, C. jejuni subsp. jejuni, clinical associations of emerging campylobacteria are strikingly similar (Fig. 3). Among pediatric clinical isolates, the percentages of C. concisus, C. upsaliensis, and C. jejuni subsp. doylei were similar to those of C. jejuni subsp. jejuni for patients with diarrhea (80 to100%), vomiting (3 to12%), and fever (4 to 9%) (1). Comparing the frequency of Campylobacter spp. isolated from pediatric blood and stool cultures (Fig. 4), the recognized pathogen C. jejuni subsp. jejuni is isolated almost equally as often as the emerging pathogen C. upsaliensis. However, another emerging Campylobacter, the infrequently isolated C. jejuni subsp. doylei, is three times more likely to be isolated from blood than stool culture. Also of note is the over-representation of Helicobacter fennelliae and H. cinaedi as blood culture isolates, implying a pathogenic, possibly invasive function for these organisms. H. fennelliae and H. cinaedi are seldom isolated, possibly because they are H2 requiring and slow growing and produce very fine colonies or even confluent growth on the culture plate, making isolation and subsequent identification extremely difficult. The literature has ample clues to the pathogenic or putative pathogenic potentials of emerging campylobacteria. Maher et al. (11) evaluated a 16S/23S PCR/DNA probe membrane-based colorimetric assay for the detection of Campylobacter species from the feces of 127 patients with symptoms of acute gastroenteritis. Using selective agar, enrichment, and incubation for 48 h at 37ºC, 18 fecal samples were culture positive for Campylobacter. The 16S/ 23S PCR/DNA probe assay detected Campylobacter DNA in 17 of the 18 culture-positive specimens and in 41 of the 109 culture-negative specimens. The presence of Campylobacter DNA in the culture-negative samples was confirmed by a16S PCR/DNA probe assay. DNA sequence analysis of seven Clinical Microbiology Newsletter 28:7,2006 Table 3. Prescreen tests for the identification of Campylobacter and related organisms O.5 M KOH DNA “string” test L-ALA testa Gram-negative bacteria + + Gram-positive bacteria – – Campylobacter/Helicobacter/ Arcobacter + – Organisms a Oxoid Biochemical Identification system (O.B.I.S.) or Fluka 75554. Table 4. Distribution of Campylobacter species and related organisms isolated from pediatric diarrheic stools at the Red Cross Children’s Hospital, Cape Town, South Africa, 1 Oct. 1990 through 31 May 2005 Species No. % C. jejuni subsp. jejuni C. concisus C. upsaliensis C. jejuni subsp. doylei H. fennelliae C. coli H. cinaedi C. hyointestinalis CLO/HLOa Arcobacter butzleri C. fetus subsp. fetus “H. rappini” C. lari C. curvus C. rectus C. sputorum bv. sputorum 1,759 1,340 1,280 417 314 159 53 52 27 19 9 5 3 2 2 2 32.30 24.63 23.52 7.70 5.76 2.93 0.97 0.96 0.48 0.36 0.16 0.09 0.06 0.04 0.04 0.04 Total 5,443 100.00 a CLO/HLO: Campylobacter or Helicobacter organisms that died before being fully characterized. of the 16S/ 23S and five of the 16S PCR products amplified from a selection of these specimens confirmed the presence of C. jejuni subsp. jejuni, C. concisus, C. curvus, and C. gracilis DNA. The finding of Campylobacter sp. DNA in a large number of fecal specimens from patients with no other identified cause of diarrhea suggests that Campylobacter sp. other than C. jejuni subsp. jejuni may account for some cases of acute gastroenteritis in which no etiological agent is identified at present. Campylobacter species found in the oral cavity have rarely been reported to cause extraoral infections. However, Han et al. (12) recently reported three cases of extraoral abscesses caused by oral Campylobacter spp. The spread was most likely by means of the lympohematogenous system. Significantly, these Campylobacter species were all isolated anaerobically and not by the conventional microaerophilic proce© 2006 Elsevier Figure 3. Diarrheal characteristics of Campylobacter infections. CC, Campylobacter concisus; CJJ, C. jejuni subsp. jejuni; CJD, C. jejuni subsp. doylei; CU, C. upsaliensis; HF, Helicobacter fennelliae. dures usually used for Campylobacter isolation. These organisms were identified by sequencing analysis of the 16S rRNA gene. The cases included a breast abscess caused by Campylobacter 0196-4399/00 (see frontmatter) 53 Conclusion New species of Campylobacter and the related genera Arcobacter and Helicobacter are being identified on a regular basis. Emerging Campylobacter species may play a much greater role in human and animal disease than has been previously recognized. Because methods originally formulated for the isolation of C. jejuni subsp. jejuni often 54 0196-4399/00 (see frontmatter) 40 Blood n = 110 35 Stool n = 2,802 30 Percent rectus in a lymphoma patient, a liver abscess caused by C. curvus in an ovarian cancer patient, and a post-obstructive bronchial abscess caused by C. curvus in a lung cancer patient. All three patients were treated with antibiotics with complete resolution of the lesions. This report is an example of the increasingly frequent clinical reports involving emerging campylobacters. C. fetus was previously considered a cause of bacteremia in elderly men with chronic underlying illness, but AIDS patients are now the most typically infected population. C. fetus, while not an emerging Campylobacter species, is definitely an under-isolated and underappreciated one. This organism may occur in unexpected sites in the body that are usually not known to be linked with bacterial infection (3). Occasional seeding to an organ may occur, which can lead to complications. C. fetus can remain latent after bacteremic seeding in a bony focus of an immunocompromised host, only to be reactivated years later. It has been responsible for infection in a postoperative prosthetic hip joint, chronic osteomyelitis of the ankle, and pyrogenic vertebral osteomyelitis (1). C. fetus exhibits an affinity for vascular tissue, and infections have been associated with cellulitis, thrombophlebitis, and mycotic aneurysms (3). The central nervous system may be affected by C. fetus, and meningoencephalitis is the most common presentation in both children and adults. Subarachnoid hemorrhages, brain abscesses, and cerebral infarctions can occur (1). Emergent campylobacters are occasionally isolated from non-enteric body sites. C. sputorum bv. sputorum is normally found in the human oral cavity and gastrointestinal tract, but it has been isolated from abscesses of the groin and perianal and axillary areas. C. upsaliensis has been isolated from a breast abscess (3). 25 20 15 10 5 0 C jj C jd C up H cin H fen Cc C co Other Figure 4. Comparative prevalences of Campylobacter spp. isolated from pediatric blood and stool cultures at the Red Cross Children’s Hospital, Cape Town, South Africa. C jj, Campylobacter jejuni subsp. jejuni; C jd, C. jejuni subsp. doylei; C up, C. upsaliensis; H. cin, Helicobacter cinaedi; H fen, H. fennelliae; C c, C. concisus; C co, C. coli; Other, various other Campylobacter spp. fail to support the growth of fastidious emergent campylobacteria, these organisms are largely under-detected in clinical specimens. Membrane filtration onto antibiotic-free media and incubation in an H2-enriched microaerobic atmosphere at 37°C is a simple, efficient, and costeffective isolation protocol for the isolation of all known Campylobacter, Arcobacter, and Helicobacter spp. and is the only method presently available for the isolation of some hydrogendependent Campylobacter spp., such as C. concisus, C. rectus, and C. curvus. Use of the Cape Town protocol also increases the yield of C. jejuni subsp. jejuni strains. At present, the roles that emergent Campylobacter spp. play in the disease process are not fully understood. This is unlikely to change until the correct protocols essential for the isolation and identification of emerging Campylobacter spp. are used in microbiology laboratories involved in the performance of routine diagnosis, surveillance, epidemiologic, and other studies. Additional research is essential for a better definition of the prevalence, persistence, and range of infection associated with these very much under-isolated and underrated organisms. What is currently known of emergent campylobacters is truly only the tip of the iceberg. Acknowledgement This research is being funded by the © 2006 Elsevier Department of Science and Technology (South Africa) as a partner in the European Commission’s Fifth Framework Programme “Quality of Life and Management of Living Resources” CAMPYCHECK Project (QLK1 CT 2002 02201). The South African Medical Research Council provided additional funding. References 1. Lastovica, A.J. and M.B. Skirrow. 2000. Clinical significance of Campylobacter and related species other than Campylobacter jejuni and C. coli, p. 89-120. In I. Nachamkin and M.J. Blaser (ed.), Campylobacter, 2nd ed. ASM Press, Washington, D.C. 2. Loades, C.J., L.E. Reiman, and C.W. Keevil. 2005. Development of an improved medium for recovery of Campylobacter species avoiding common selective antibiotics, abstr. D-198. In Abstr. 105th Gen. Meet. Am. Soc. Microbiol. American Society for Microbiology, Washington, D.C. 3. Lastovica, A.J., M.E. Engel, and M.J. Blaser. 2002. Atypical Campylobacters and related microorganisms, p. 741-761. In M.J. Blaser et al. (ed.), Infections of the gastrointestinal tract, 2nd ed. Lippincott Williams & Wilkins, New York. 4. Le Roux, E. and A.J. Lastovica. 1998. The Cape Town Protocol: how to isolate the most Campylobacters for your dollar, pound, franc, yen, etc., p. 30-33. In A.J. Lastovica, D.G. Newell, and E.E. Clinical Microbiology Newsletter 28:7,2006 Lastovica (ed.), Campylobacter, Helicobacter and related organisms. Institute of Child Health, Cape Town, South Africa. 5. Moore J.E. et al. 2005. Campylobacter. Vet. Res. 36:351-382. 6. Sylvester, F.A. et al. 1996. Adherence to lipids and intestinal mucin by a recently recognised human pathogen, Campylobacter upsaliensis. Infect. Immun. 64:4060-4066. 7. Fouts, D.E. et al. 2005. Major structural differences in novel potential virulence mechanisms from genomes of multiple campylobacter species. PLOS Biol. 3(1)e15. 8. Pickett, C.L. et al. 1996. Prevalence of cytolethal distending toxin production in Campylobacter jejuni and relatedness of Campylobacter sp. cdtB gene. Infect. Immun. 64:2070-2078. 9. Alam, K. et al. 2005. Direct comparison of ICDDR, B and Cape Town Protocols for the isolation and characterization of Campylobacter species. A1, p. 2. In Abstr. CHRO 2005. 13th Int. Workshop Campylobacter, Helicobacter and related organisms. Griffith University, Queensland, Australia. 10. Hoosain, N. and A.J. Lastovica. 2005. Evaluation of the Oxoid Biochemical Identification System (O.B.I.S.) for the differentiation of Campylobacter and Arcobacter from other gram-negative organisms. A36, p. 13. In Abstr. CHRO 2005. 13th Int. Workshop Campylobacter, Helicobacter and related organisms. Griffith University, Queensland, Australia. 11. Mahler, M. et al. 2003. Evaluation of culture methods and a DNA probe-based PCR assay for the detection of Campylobacter species in clinical specimens of feces. J. Clin. Microbiol. 41:2980-2986. 12. Han, X.Y., J.J. Tarrand, and D.C. Rice. 2005. Oral Campylobacter species involved in extraoral abscess: a report of three cases. J. Clin. Microbiol. 43:2513-2515. Call for Submissions Case Reports If your laboratory has isolated an uncommon organism, a common organism from an unusual patient, or an organism that presented a particular diagnostic challenge, why not share the information with your colleagues through the Clinical Microbiology Newsletter. The editors would like to receive interesting case reports from our readers for possible publication in the Newsletter. Submitted case reports should contain (i) a brief clinical history summarizing the symptoms and course of the illness, (ii) a description of how the organism(s) was cultured and differentiated from closely associated organisms, and (iii) the results of susceptibility tests for the isolate(s). Letters Letters expressing opinions or offering helpful technical hints will be considered for publication (subject to editing) provided they are signed by all authors and do not exceed two typewritten (double-spaced) pages. Preparation and Submission of Material All material submitted for publication in the Newsletter (including references) should be typed double-spaced, with 1inch margins, on standard 8 1/2 × 11-inch paper. Photographic prints should be in black and white, not color. Charts and graphs should also be sent as black-andwhite hardcopy. If you submit a review article, please include a short abstract Clinical Microbiology Newsletter 28:7,2006 of not more than 150 words. If you prepared your manuscript on a personal computer, please also send the manuscript on diskette and indicate the program used to create it. Electronic files for charts and graphs should be submitted as separate files in EPS, TIFF, or JPEG format. Please include your phone and FAX numbers and e-mail address. References Authors are responsible for the accuracy of references, which should include complete publication information. References should be listed in the numerical order by which they occur throughout the text, numbered consecutively, and cited in the text by these numbers. List the first author in a reference with last name followed by initials; list subsequent authors in the same reference with initials followed by last names. (Editors are always listed with initials followed by last names.) If there are more than three authors, include only the name of the first author followed by “et al.” Include abstracts, materials in press, personal communications, and submitted material in the references section. Citations to personal communications must be accompanied by a letter from the person cited giving permission for the citation. Some typical examples of references are listed below. 1. Petts, D.N. 1999. Evaluation of the Oxoid Dryspot streptococcal grouping kit for grouping beta-hemolytic strepto© 2006 Elsevier cocci. J. Clin. Microbiol. 37:255-257. 2. Reich, K.A. and G.K. Schoolnik. 1996. Halovibrin, secreted from the light organ symbiont Vibrio fischeri, is a member of a new class of ADP-ribosyltransferases. J. Bacteriol. 178:209-215. 3. Walls, J.J., K.M. Asanovich, and J.S. Dumler. 1998. Comparison of Ehrlichia equi and human granulocytic ehrlichiosis (HGE) agent strains for IFA serodiagnosis of HGE. C-208, p. 165. In Abstracts of the 98th General Meeting of the American Society for Microbiology 1998. ASM Press, Washington, D.C. 4. McGough, D.A. et al. 1994. Fungi and fungal infections, p. 1169-1196. In K.D. McClatchey (ed.), Clinical laboratory medicine. Williams & Wilkins, Baltimore, MD. 6. Bennish, M.L. 1994. Cholera: pathophysiology, clinical features, and treatment, p. 229-255. In I.K. Wachsmuth, P.A. Blake, and O. Olsvik (ed.), Vibrio cholerae and cholera, molecular to global perspectives. ASM Press, Washington, D.C. 5. Jones, T. Personal communication. Address for Submissions Send the original typescript (double spaced, four copies), diskette, and any photographs and figures to: Paul A. Granato, Ph.D. Dept. of Microbiology & Immunology WH 2204 SUNY Upstate Medical University 750 E. Adams St. Syracuse, NY 13210 USA 0196-4399/00 (see frontmatter) 55 Erratum ... Susceptibility Report (S = Susceptible, I = Intermediate, R = Resistant)......... Clinical Microbiology Newsletter, February 15, 2006 issue, Vol. 28, No.4, p. 28 An error occurred in the reproduction of Figure 4 that appeared in the article authored by Dr. Joan Barenfanger entitled “Quality Assurances: Decreasing Clinically Irrelevant Testing from Clinical Microbiology Laboratories, Part II” that was published in CMN vol. 28, no.4, p.28. The original format of Figure 4 appears to the right. The original intent was to show how poorly the example report was configured. The published figure was incorrectly formatted to make the report appear clear and easily comprehensible. S AUREUS P AERUG MIC INTERP KB AZTREONAM S CEFEPIME S LEVOFLOXACIN S CEFTAZIDIME S CIPROFLOXACIN S ERYTHROMYCIN R GENTAMICIN I IMIPENEM OXACILLIN S S PIP/TAZOBACTAM PENICILLIN G TOBRAMYCIN S R S Figure 4. Replica of actual report configuration of a major LIS vendor in a hospital. 56 0196-4399/00 (see frontmatter) © 2006 Elsevier Clinical Microbiology Newsletter 28:7,2006