Association Study Between an Outbreak of Guillain-Barre Syndrome in Jilin,China,and Preceding Campylobacter jejuni Infection

Abstract

From June to July 2007, 36 cases of Guillain-Barre syndrome (GBS) occurred in a township in north China. Serological study and bacteria culture were performed to investigate the association between preceding Campylobacter jejuni infection and this GBS outbreak. Anti–C. jejuni antibodies were found in significantly higher numbers of GBS patients (IgM 84%, IgG 87.5%) than in healthy inspection cases (IgM 33%, IgG 27%). IgG anti-GM1 was the dominant anti-ganglioside antibody among the GBS patients. Seven C. jejuni isolates (four from human stool and three from poultry specimens taken from the patients' houses) were obtained. Serotyping and molecular analysis were used to investigate the genetic relatedness among these C. jejuni isolates. The four human isolates, collected from residents of the same district, were indistinguishable by both pulsed-field gel electrophoresis and multilocus sequence typing, suggesting these patients had a common source of infection. A new sequence type, sequence type-2993, was assigned to the human C. jejuni isolates, three of which belonged to Penner serotype heat-stable (HS):41. Both serotype and molecular subtype of the human C. jejuni isolates were different from those of isolates obtained from poultry specimens. Our results suggest that the antecedent C. jejuni infection triggered this GBS outbreak in China.

Introduction

W ith the near complete elimination of poliomyelitis, Guillain-Barre syndrome (GBS) is the most common cause of acute flaccid paralysis (AFP) in the world with annual incidence ranging from 0.4 to 4 GBS cases per 100,000 of the population (Hughes and Rees, 1997; Hahn, 1998). Although most cases have an uneventful recovery, 15% to 20% of GBS patients suffer from neurological deficits. The mortality rate ranges from 3% to 15% in both developed and developing countries (Hughes and Rees, 1997; Fletcher et al., 2000; Kuwabara, 2004). Several subtypes of GBS exist, including a demyelinating subtype, namely acute inflammatory demyelinative polyneuropathy (AIDP), which is reported predominately in the United States and in Europe; and acute motor axonal neuropathy (AMAN), the predominant form in China and in Mexican children (Ho et al., 1995; Kuwabara, 2007; Nachamkin et al., 2007). A number of antecedent viral and bacterial infections have been linked to GBS, Campylobacter jejuni infection being most commonly identified. The induction of anti-ganglioside antibodies by molecular mimicry between the cell wall and lipopolysaccharides of C. jejuni and the glycolipids in peripheral nerve has been shown to play a crucial role in the pathogenesis of Campylobacter-associated GBS (Nachamkin, 2001; Perera et al., 2007).

The reported incidence of GBS in China is about 0.67–1.68 cases per 100,000, and reports from the National AFP case surveillance system in China show that GBS is the predominant cause of AFP among youths (under 15 years old) (Zhang et al., 1994, 2004, 2006). According to national surveillance data, diarrheal illness occurs most frequently in the summer months in China, with outbreaks of C. jejuni infection infrequently reported (Wang and Gan, 2003; Xu and Shen, 2006). A previous study suggested that the yearly summer epidemic of GBS in the rural area of Hebei province in north China was linked to C. jejuni infections (Ho et al., 1995). From June 29 to July 5, 2007, the local AFP case surveillance system in Changchun, Jilin, notified China CDC of a GBS outbreak involving 30 cases. By July 23, an additional six cases were reported. A total of 36 GBS patients, all from the Shuangyang district, Changchun, Jilin, were identified. During the retrospective field investigation conducted in response to the GBS outbreak, the number of diarrheal cases reported in the Shuangyang district between June 19 and July 2, 2007, increased and was higher than in neighboring districts. During the weeks of June 19 to June 25 and June 26 to July 2, the number of diarrheal cases in Shuangyang district were 63 and 54, respectively. These were 3.15 and 6.75 folds higher than during the same time period in 2006 in this district. The incidence of diarrhea in Shuangyang (780/100,000) between June 19 and July 2, 2007, was 11.38 and 12.68 folds higher than in its neighboring districts of Nongan (63/100,000) and Jiutai (57/100,000) (unpublished data). The high number of diarrheal cases in Shuangyang coincided with the replacement in June of the district's old water supply system, which had dried up. Four 1 L water samples taken from the new water supply system all tested negative for Campylobacter by culture and polymerase chain reaction (PCR) (unpublished data). The aim of this study was to investigate the association of the GBS cases identified in the Shuangyang district with preceding enteric infection caused by C. jejuni.

Materials and Methods

Patients

The study population included 32 patients with clinically defined GBS admitted to three hospitals in Changchun between June 23 and July 9. GBS cases were defined based on the National Institutes of Neurological and Communicative Disorders and Stroke (NINCDS) criteria (Asbury et al., 1978) and reviewed by clinical experts according to clinical outcome, cerebrospinal fluid examination, and electromyogram test. Subclassification for AIDP and AMAN was based on clinical characteristics and electromyogram diagnosis. Characteristics of the 32 GBS cases are listed in Table 1. All 32 GBS patients were from the Shuangyang district, a township in the provincial city of Changchun, Jilin province, China.

Table 1.

Characteristics of 32 Guillain-Barre Syndrome Cases, Changchun, China, 2007

	Clinical diagnosis
	AMAN	AIDP	Not classified
Number	15	3	14^a
Male/female	11/4	2/1	8/6
Age median	20	26	20
Respiratory support	27% (4/15)	0	7.14% (1/14)
Diarrhea	60% (9/15)	33% (1/3)	78.6% (11/14)
Seropositive for Campylobacter jejuni	IgM: 100% (15/15) IgG: 100% (15/15)	IgM: 67% (2/3) IgG: 67% (2/3)	IgM: 71% (10/14) IgG: 78.6% (11/14)
Stool culture positive for C. jejuni	1	0	0

This number included one case that did not belong to AMAN or AIDP.

AIDP, acute inflammatory demyelinative polyneuropathy; AMAN, acute motor axonal neuropathy.

Serologic study

One hundred and eleven serum samples were collected from 95 human subjects, of which 32 were GBS patients, 33 were hospital-registered diarrheal cases in Shuangyang district selected between June 23 and July 14, and 30 were healthy controls selected randomly from a local health inspection center in the district with age and sex matched to GBS patients. The healthy controls were selected over a period from January to June 2007. They did not have any underlying illness and were attending the health inspection center for regularly scheduled health checkups. Sera were collected from the 32 hospitalized GBS patients on admission (between June 23 and July 9), and a second set of sera was collected 10–14 days later from the 16 patients remaining in the hospital. Serologic tests to determine IgM and IgG to C. jejuni were performed for all 111 samples using an ELISA previously standardized in our laboratory (Li et al., 1993). The same serological tests were performed on the intravenous immunoglobulins used for the GBS patients' therapy in the hospital. Prevalence of IgM and IgG against ganglioside GM1, Asialo GM1, GD1a, GD1b, and GQ1b in serum was measured using a commercial ELISA kit (IMMCO Diagnostics) for 89 subjects: 32 GBS patients (first-phase collection), 30 diarrheal cases, and 27 healthy controls. The sera were coded and assayed blind twice on separate occasions.

Bacteria isolation and identification

Human stool specimens were collected from 78 subjects for Campylobacter testing. Twenty-seven were patients with clinical features of GBS, admitted to the same hospital in Changchun city, and 51 were either household relatives (n = 12) or neighbors of these patients (n = 39). Stool specimens and medical histories of these 51 subjects were obtained at their houses by China CDC personnel during the retrospective field investigation. Seven of them had current diarrhea, 34 had recent diarrheal history, and 10 had no diarrhea within the last 30 days. Seventeen poultry fecal samples were collected from 13 chickens and 4 geese raised in the houses of 5 GBS patients. All human and poultry stool samples were preenriched in Preston selective broth and then subinoculated on Campylobacter selective medium (Columbia Agar Base; Oxoid CM0331, supplemented with 5% of sheep blood and Campylobacter selective supplement; Oxoid SR0117) and incubated at 42°C in a microaerophilic atmosphere (5% O₂, 10% CO₂, and 85% N₂) for 72 h. The primary culture plates were checked every day and discarded 10 days later. Suspected Campylobacter colonies were characterized by Gram stain, oxidase and catalase tests, and API Campy identification system test (API, REF 20800). Campylobacter species-specific PCR for C. jejuni, C. coli, C. lari, C. upsaliensis, and C. fetus subsp. fetus were performed according to previous reports (Denis et al., 1999; Wang et al., 2002; Klena et al., 2004).

Penner serotyping

C. jejuni isolates recovered during this study were serotyped by the heat-stable (HS) serotyping scheme of Penner and Hennessy using a commercial 25 Penner heat-stable antisera set (Campylobacter Antisera Seiken Set; Denka Seiken) previously described (Rautelin and Hanninen, 1999). In addition to the negative control in the kit, two C. jejuni strains were selected as reference strains: C. jejuni strain ATCC 700819 (Penner serotype HS:2) and one Chinese C. jejuni strain (Penner serotype HS:19).

Pulsed-field gel electrophoresis and multilocus sequence typing analysis

Preparation of C. jejuni DNA, macrorestriction analysis using the restriction enzymes SmaI and KpnI, and pulsed-field gel electrophoresis (PFGE) were performed according to the standard PulseNet PFGE protocol for C. jejuni (www.cdc.gov/pulsenet/protocols/campy_protocol.pdf). PFGE and cluster analysis were performed using BioNumerics (Version 4.1; Applied Maths) and the dice coefficient using the unweighted pair group method with arithmetic averages. Isolates were assigned to the same PFGE macrorestriction profile when they clustered at greater than 95% similarity (for SmaI, 0.45% optimization and 0.85% position tolerance; for KpnI, 0.41% optimization and 1.50% position tolerance).

Multilocus sequence typing (MLST) was performed by sequencing seven housekeeping gene loci (aspA, glnA, gltA, glyA, pgm, tkt, and uncA) using previously described primers (Dingle et al., 2001). Briefly, each of the seven gene fragments was amplified by PCR with Pyrobest DNA polymerase (TaKaRa Bio), purified, and sequenced by an ABI Prism 377XL DNA Sequencer (Applied Biosystems). The sequence data for each of the seven MLST loci were compared with sequences in the MLST database (www.pubmlst.org/campylobacter) to determine the allele number. The sequence type (ST) of each strain was assigned from the profiles of the seven alleles in the MLST database.

Antibiotic susceptibility test

Minimal inhibitory concentrations (MIC) for 10 antibiotics, tetracycline, nalidixic acid, ciprofloxacin, levofloxacin, metronidazole, gentamicin, streptomycin, erythromycin, chloramphenicol, and ampicillin, were determined on Campylobacter isolates using the E-test system (AB Biodisk). The quality control strain used was C. jejuni ATCC 33560.

Statistical analysis

Differences in frequency of positive results for the antibodies to C. jejuni and to various gangliosides between GBS patients and the healthy inspection cases were tested with the chi-square test.

Results

Clinical details

Thirty-two confirmed cases of GBS were identified among residents of Shuangyang during June to July 2007. Subclassification was performed for 19 of the 32 cases within 2 weeks of the patients' admission to hospital. The personal, clinical, and laboratory characteristics of the outbreak-associated cases are shown in Table 1. Of the 32 GBS patients, 66% (21/32) experienced diarrheal symptoms during the previous 3–40 days; the median period was 10 days before the onset of GBS symptoms.

Serologic testing

The positive ratios and the statistical analysis results for the antibodies to C. jejuni are shown in Table 2. Anti–C. jejuni antibodies were not found in the intravenous immunoglobulins used to treat the GBS patients. Twenty-seven (84%, 27/32) GBS patients, 15 of whom were diagnosed with AMAN, 2 with AIDP, and 10 with unclassified type, were sera IgM positive. Twenty-eight (87.5%, 28/32) GBS cases were IgG positive (15 AMAN, 2 AIDP, and 11 unclassified) against C. jejuni. Twenty-six GBS patients were both IgM and IgG positive against C. jejuni compared to 3 cases with both IgM and IgG positive in the healthy inspection cases group. The frequency of anti–C. jejuni antibodies among GBS patients was significantly higher than in both the diarrheal cases group (IgM: χ ² = 21.44, p < 0.005; IgG: χ ² = 14.44, p < 0.005) and the healthy inspection cases group (IgM: χ ² = 16.76, p < 0.005; IgG: χ ² = 23.53, p < 0.005). No significant difference was found in the frequency of anti–C. jejuni antibodies in either the two sets of serum specimens taken 10–14 days apart (IgM: χ ² = 0.08, p > 0.05; IgG: χ ² = 1.2, p > 0.05) or between the diarrheal group and the healthy inspection cases group, both with regard to IgG and IgM (Table 2).

Table 2.

Anti–Campylobacter jejuni Antibody Prevalence in Sera

	Seropositive ratio
Source	IgM	IgG	Chi-square test (compared with control)
GBS cases (first-phase collection)	84% (27/32)	87.5% (28/32)	IgM: χ ² = 16.76; p < 0.005 IgG: χ ² = 23.53; p < 0.005
GBS cases (second-phase collection)	87.5% (14/16)	75% (12/16)	IgM: χ ² = 12.27; p < 0.005 IgG: χ ² = 9.92; p < 0.005
Diarrhea cases	27% (9/33)	42% (14/33)	IgM: χ ² = 0.27; p > 0.5 IgG: χ ² = 1.72; 0.25 > p > 0.1
Healthy inspection cases	33% (10/30)	27% (8/30)	As control group

One hundred and eleven serum samples were divided into four groups. The healthy inspection cases group was set up as the control group. All the samples were tested by the same lot number kit. The tests for the two phases of collection for GBS patients were performed using the same kit.

GBS, Guillain-Barre syndrome.

Anti-glycolipid serology

The frequency of anti-ganglioside antibodies in GBS patients and controls is shown in Table 3. IgG anti-GM1 and IgG anti-GD1b were the dominant anti-gangliosides among the GBS patients. From chi-square test, IgG specificity to GMl (69%), Asialo GM1 (34%), and GD1b (53%) antibodies were significantly higher in GBS patients than in controls (χ ² = 18, p < 0.005; χ ² = 11.7, p < 0.005; χ ² = 11.9, p < 0.005). Additionally, among the GBS patients, 71% (20/28) of the C. jejuni seropositive cases were anti-GM1 IgG positive, 36% (10/28) were anti-Asialo GM1 IgG positive, and 61% (17/28) were anti-GD1b IgG positive. Seventeen of the 28 (61%) C. jejuni seropositive GBS cases had multiple anti-ganglioside antibodies. The C. jejuni seropositivities among the anti-GM1, anti-Asialo GM1, and anti-GD1b IgG positive groups were 91% (20/22), 91% (10/11), and 100% (17/17), respectively. The frequencies of antibodies to GD1a and GQ1b were significantly lower than for antibodies to GM1 and GD1b among GBS patients.

Table 3.

Prevalence of Anti-Ganglioside Antibodies in Sera

	Seropositive ratio
Anti-ganglioside antibodies	GBS cases (32)	Diarrhea cases (30)	Healthy inspection cases (27)
Anti-GM1 IgM	12.5% (4/32)	3% (1/30)	4% (1/27)
Anti-GM1 IgG	69% (22/32)	23% (7/30)	22% (6/27)
Anti-Asialo GM1 IgM	6% (2/32)	3% (1/30)	4% (1/27)
Anti-Asialo GM1 IgG	34% (11/32)	3% (1/30)	7% (2/27)
Anti-GD1a IgM	0	0	0
Anti-GD1a IgG	16% (5/32)	10% (3/30)	0
Anti-GD1b IgM	12.5% (4/32)	3% (1/30)	4% (1/27)
Anti-GD1b IgG	53% (17/32)	3% (1/30)	0
Anti-GQ1b IgM	6% (2/32)	0	0
Anti-GQ1b IgG	19% (6/32)	0	0

Eighty-nine serum samples from 89 subjects (32 GBS patients, 30 diarrhea cases, and 27 healthy inspection cases) were picked for the anti-ganglioside test.

Microbiology

Fourteen Campylobacter isolates were obtained from stool culture after 3 days. Based on phenotypic characterization and species-specific PCR, seven were identified as C. jejuni and seven as C. coli. Four of the seven C. jejuni isolates were recovered from human stool and three from chicken fecal samples. The characteristics of these seven C. jejuni isolates are summarized in Table 4. Isolate ICDCCJ07001 was obtained from a GBS patient who had machine ventilation for 180 days and was both IgM and IgG positive against C. jejuni in serum. Isolates ICDCCJ07002, ICDCCJ07003, and ICDCCJ07004 were all from the neighbors of GBS patients, none of whom were hospitalized. One of these individuals had chronic diarrhea for 21 days (ICDCCJ07002), one had recent diarrheal history (ICDCCJ07003), and one had no history of diarrhea (ICDCCJ07004) within 30 days. All these three neighbors were IgG positive and two of them (ICDCCJ07002 and ICDCCJ07004) were IgM positive against C. jejuni in serum. These four individuals from whom the C. jejuni isolates were recovered lived in geographically separate areas in the same district and shared the same water source in their houses. Three of the human C. jejuni isolates were identified as HS:41 and the remaining isolate (ICDCCJ07004) was untypeable using the commercial panel of 25 Penner heat-stab-le antisera set. Different serotypes were identified among the chicken isolates: CJHENLIU-1 was HS:21, CJHENYIN was HS:31, and CJHENLIU-2 was untypeable using the commercial serotyping kit (see Table 4). Among the C. coli isolates, one was from a neighbor of a GBS patient without recent diarrheal history and six were from the poultry (one from a goose and five from chickens).

Table 4.

Summary of Subtyping Results for the Seven Campylobacter jejuni Isolates Included in This Study

Isolate	Source	Disease	Penner serotype	PFGE SmaI mrp	PFGE KpnI mrp	ST
ICDCCJ07001	Human stool	GBS	HS:41	DBRS16.0128	DBRK02.0009	ST-2993
ICDCCJ07002	Human stool	Diarrhea	HS:41	DBRS16.0128	DBRK02.0009	ST-2993
ICDCCJ07003	Human stool	Diarrhea	HS:41	DBRS16.0128	DBRK02.0009	ST-2993
ICDCCJ07004	Human stool	Asymptomatic	NT^a	DBRS16.0128	DBRK02.0009	ST-2993
CJHENLIU-1	Chicken fecal	—	HS:21	DBRS16.1068	DBRK02.0019	ST-3927
CJHENLIU-2	Chicken fecal	—	NT^a	DBRS16.1068	DBRK02.0019	ST-2274
CJHENYIN	Chicken fecal	—	HS:31	DBRS16.1064	DBRK02.0035	ST-3924

Not typeable using commercial 25 Penner heat-stable antisera set.

mrp, macrorestriction profile; PFGE, pulsed-field gel electrophoresis; ST, sequence type.

Molecular subtyping

All four human C. jejuni isolates had indistinguishable SmaI and KpnI PFGE profiles, and they were different from the PFGE profiles of the chicken isolates (Fig. 1). Two chicken isolates from different chickens recovered from the same household had indistinguishable PFGE patterns. The human and chicken PFGE profiles were compared to previously generated PFGE data on clinical Chinese C. jejuni strains (55 SmaI patterns for 99 C. jejuni isolates and 41 KpnI patterns for 49 C. jejuni isolates, which include 8 GBS-associated isolates from north China, unpublished) by BioNumerics software (Applied Maths), and no indistinguishable patterns were identified. The PFGE patterns of the four human isolates were also compared to the PulseNet USA National Campylobacter database, and no indistinguishable patterns were identified. The unique human SmaI and KpnI patterns were assigned DBRS16.0128 and DBRK02.0009, respectively, by PulseNet USA (see Table 4). By MLST all four human C. jejuni isolates had the same ST. This ST was new in the public MLST database and assigned ST-2993, which belongs to clonal complex ST-362. The three chicken C. jejuni isolates were ST-3927, ST-2274, and ST-3924.

FIG. 1.

Pulsed-field gel electrophoresis patterns of seven Campylobacter jejuni isolates digested by SmaI (a) and KpnI (b). Lanes 1,5, and 10, PulseNet molecular size standard; Salmonella enterica serotype Braenderup H9812 (ATCC BAA-664); lane 2, isolate CJHENLIU-1; lane 3, isolate CJHENLIU-2; lane 4, isolate CJHENYIN; lane 6, isolate ICDC07002; lane 7, isolate ICDC07003; lane 8, isolate ICDC07001; lane 9, ICDC07004.

Drug susceptibility

All of the Campylobacter isolates (14/14) were resistant to tetracycline, nalidixic acid, ciprofloxacin, and levofloxacin (MIC ≥16 μg/mL). Eighty-six percent (12/14, including seven C. jejuni and five C. coli) were resistant to metronidazole (MIC ≥1 μg/mL). The four human C. jejuni isolates were highly resistant to metronidazole (MIC ≥256 μg/mL). Fifty-seven percent (8/14, three C. jejuni and five C. coli) of the isolates were resistant to ampicillin (MIC ≥16 μg/mL), 86% (12/14, except two C. coli isolates) were sensitive to streptomycin (MIC ≥16 μg/mL), and 93% (13/14, except one C. coli isolate) were sensitive to erythromycin (MIC ≥8 μg/mL) and chloramphenicol (MIC ≥16 μg/mL). All 14 isolates were sensitive to gentamicin (MIC ≤4 μg/mL).

Discussion

In the absence of positive culture, serologic evidence is one of the most reliable demonstrations for the association between GBS and antecedent C. jejuni infection (Rees et al., 1995a, 1995b; Nachamkin et al., 1998). The present study provides evidence of a link between antecedent C. jejuni infection and a large outbreak of GBS that occurred in early summer 2007 in China. Based on serological studies, up to 84% (IgM) and 87.5% (IgG) of GBS patients identified in this outbreak were seropositive to C. jejuni, compared to 33% and 27% of controls, retrospectively. This is consistent with a recent C. jejuni infection among the GBS patients. No significantly different seropositive ratio was found between the two phases of collection (10–14 days apart) for the GBS patients. The reason may be that the time interval was not long enough to change the titer of the antibodies. Further study is needed to investigate the relationship between clinical variation and antibody titers by additional serum sample collection and antibody testing. In this study, 34% (11/32) of the GBS patients had no preceding recognizable diarrhea, but 91% (10/11) of those without diarrhea had seropositive results with anti–C. jejuni antibodies. These results are consistent with GBS patients showing evidence of asymptomatic infections (Obi and Coker, 1989; Tam et al., 2006).

Many previous studies have shown that antibodies to various gangliosides play an important role in the pathophysiology of GBS and its variants (Gibbs et al., 1993; Press et al., 2001; Yuki and Kuwabara, 2007). Anti-GM1, GD1a, GM1b, and GalNAc–GD1a, each having a particular severe form of GBS, have been associated with a preceding C. jejuni infection (Rees et al., 1995a; Jacobs et al., 1996; Yuki et al., 1999; Nachamkin et al., 2002). Previous studies in China showed that C. jejuni is an important pathogen that caused a peak of GBS cases in early summer in northern China, AMAN being the main form (Griffin et al., 1995; Ho et al., 1995). The C. jejuni strain HB93-13 (HS:19) isolated from a GBS patient in northern China was shown to express GM1 and GD1a ganglioside-like structures (Rees et al., 1995a). In the present study, although only 19 GBS cases were subclassified, 15 of them belonged to AMAN, all of which were both IgM and IgG seropositive against C. jejuni. AMAN appears to be the dominant subtype of this GBS outbreak also. The serotype of the isolates from the GBS patient and the two diarrheal neighbors in this study was HS:41, which has previously been shown to be a highly clonal serotype and to be overrepresented among GBS-associated strains in South Africa (Goddard et al., 1997; Lastovica et al., 1997). IgG anti-GMl (69%) was the predominant anti-ganglioside antibody among the GBS patients in our present study, which is consistent with a previous report that C. jejuni serotype HS:41 LOS and the GM1 ganglioside have similar epitopes (Prendergast et al., 1998). Our study reported the first HS:41 C. jejuni strain isolated from both GBS patients and diarrheal cases in China.

Molecular subtyping including PFGE and MLST has previously been shown to be useful for investigating Campylobacter outbreaks (Dingle et al., 2001, 2002; Fitzgerald et al., 2001; Olsen et al., 2001). In this study, both PFGE and MLST analysis indicated that the four human C. jejuni isolates were genetically indistinguishable from each other, suggesting a common source of infection, but different from the household chicken C. jejuni strains. The retrospective field investigation had suspected the replacement of the water system in the Shuangyang district as one possible source of the human Campylobacter infection, because the number of diarrheal cases in the area increased at the time the water system was replaced and the GBS patients were from the region where the water system was changed. However, by the time environmental samples were taken, the suspected new water system had been disinfected, which may help explain why the four water samples all tested negative for Campylobacter by culture and PCR.

The PFGE SmaI and KpnI profiles of the GBS strain were unique when compared to 5284 entries in the PulseNet USA National Campylobacter database. The PFGE profiles were most closely related to those of four C. jejuni HS:41 isolates associated with an outbreak in Louisiana in 1995. This high level of similarity (80%) is not unexpected given the reported clonal nature of this serotype. Searching in the MLST database showed that there were three STs (four isolates) that shared six of seven identical allele profiles as ST-2993 (see Table 5). Isolates 82/12008 and 82/13825 were obtained from the blood of systemic disease patients in UK. Isolate RM3148 belonged to serotype HS:41 and was isolated from a GBS patient in Mexico.

Table 5.

Characteristics of Campylobacter jejuni Isolates from the Multilocus Sequence Typing Database That Have at Least Six Identical Sequence Type Alleles as Sequence Type-2993

Isolate	Country	Disease	Source	Penner	ST	aspA	glnA	gltA	glyA	pgm	tkt	uncA	Clonal complex
ICDCCJ07001	China	GBS	stool	HS:41	2993	1	2	42	4	11	9	8	ST-362
82/12008	UK	Systemic disease	blood	HS:46	27	1	2	42	85	11	9	8	ST-362
82/13825	UK	Systemic disease	blood	ND	27	1	2	42	85	11	9	8	ST-362
H78409	UK	Gastroenteritis	stool	ND	1377	1	2	42	4	153	9	8	ST-42
RM3148	Mexico	GBS	stool	HS:41	1672	1	2	42	4	256	9	8	ST-42

ND, not determined.

The antibiotic susceptibility test results showed that all the isolates, both from human and poultry, were coresistant to tetracycline, nalidixic acid, ciprofloxacin, and levofloxacin. This explains why the patient with persistent diarrhea for 21 days did not recover despite taking levofloxacin for home medication. Comparison with other recent susceptibility testing results from our laboratory showed that 100% (30/30) of C. jejuni strains from diarrheal patients isolated from 2006 to 2007 were resistant to fluoroquinolone (unpublished data). Therefore, the selective pressure of the antibiotics for C. jejuni strains in the environment needs to be considered further in China.

Although the pathogen plays a role in determining if a C. jejuni infection will develop into GBS, host susceptibility factors are also involved in susceptibility to the development of GBS (Rees et al., 1995c; Monos et al., 1997). Such factors might explain why in this study only some patients developed GBS and different subtypes of GBS were observed, even though other diarrheal cases among neighbors of the GBS patients had the same outbreak strain. Genetic analysis of the pathogen and background investigation of the host would be helpful in the future to futher elucidate this.

Conclusions

The serology, microbiology, and molecular subtyping results described in this study suggest that a preceding C. jejuni infection triggered this largest reported GBS community outbreak in China, and C. jejuni ST-2993 (HS:41) was the epidemic C. jejuni strain. The results place emphasis on the necessity and urgency to explore how much GBS is attributable to Campylobacter and the pathogen active surveillance in China. Further work is ongoing to investigate the virulence potential of this specific C. jejuni strain.

Footnotes

Acknowledgments

This work was supported by grants from the National Key Program for Infectious Disease of China (2008ZX10004-002, 2008ZX10004-008, and 2008ZX10004-009) and the Research for Major Food-Borne Pathogen Tracking Technique from the Ministry of Science and Technology of the People's Republic of China (2006BAK02A15).

Disclosure Statement

No competing financial interests exist.

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