Potilaista eristettyjen kampylobakteerikantojen tunnistus ja epidemiologinen tyypitys

C. jejuni constitutes the majority of Campylobacter strains isolated from patients in Finland, and C. coli strains are also reported. To improve the species identification, a combination of phenotype- and genotype-based methods was applied. Standardising the cell suspension turbidity in the hippurate hydrolysis test enabled the reliable identification of hippurate-positive Campylobacter strains as C. jejuni. The detection of species-specific genes by PCR showed that about 30% of the hippurate-negative strains were C. jejuni. Three typing methods, serotyping, PCR-RFLP analysis of LOS biosynthesis genes and pulsed-field gel electrophoresis (PFGE) were evaluated as epidemiological typing tools for C. jejuni. The high number of non-typeable strains lowered the discriminatory ability of serotyping. PCR-RFLP typing offered high discrimination for both serotypeable and non-typeable strains, but the correlation between serotypes and RFLP-types was not high enough to enable its use for molecular serotyping of non-typeable strains. PFGE was a highly discriminative typing method. Although the use of two restriction enzymes generally increases the discriminatory ability, KpnI alone offered almost as high discrimination as the use of SmaI and KpnI. The characteristic seasonal distribution of Campylobacter infections with a peak in summer and low incidence in winter was mainly due to domestically acquired infections. Of the C. jejuni strains, 41% were of domestic origin compared to only 17% of the C. coli strains. Serotypes Pen 12, Pen 6,7 and Pen 27 were significantly associated with domestic C. jejuni infections, Pen 1,44, Pen 3 and Pen 37 with travel-related infections. Pen 2 and Pen 4-complex were common both in domestic and travel-related infections. Serotype Pen 2 was less common among patients 60 years or older than in younger patients, more prevalent in Western Finland than in other parts of the country and more prevalent than other serotypes in winter. The source of Pen 2 infections may be related to cattle, since Pen 2 is the most common serotype in isolates from Finnish cattle. PFGE subtypes among isolates from patients and chickens during the summer 2003 and from cattle during the whole year were compared. The analysis of indistinguishable SmaI/KpnI subtypes suggested that up to 31% of the human infections may have been mediated by chickens and 19% by cattle. Human strains isolated during two one-year sampling periods were studied by PFGE. Of the domestic strains, 69% belonged to SmaI subtypes found during both sampling periods. Four SmaI subtypes accounted for 45% of the domestic strains, further typing of these subtypes by KpnI revealed six temporally persistent SmaI/KpnI subtypes. They were only occasionally identified in travel-related strains, and therefore, can be considered to be national subtypes. Each subtype was associated with a serotype: Pen 2, Pen 12, Pen 27, Pen 4-complex, Pen 41, and Pen 57. Five of these subtypes were identified in cattle (S5/K27, S7/K1, S7/K2, S7/K5 and S64/K19), and two in chickens (S7/K1 and S64/K19) with a temporal association with human infections in 2003. Cattle are more likely potential sources of these persistent subtypes, since long-term excretion of Campylobacter strains by cattle has been reported.Kampylobakteeri on yleisin suolistoinfektioita aiheuttava bakteeri. Suomessa valtakunnalliseen tartuntatautirekisteriin ilmoitetaan vuosittain 3 000 4 000 tartuntaa. Yleisin potilaista eristetty laji on C. jejuni. Myös C. coli -löydöksiä ilmoitetaan jonkin verran. Lajien tunnistusta parannettiin käyttämällä kampylobakteerin ilmiasuun (fenotyyppi) ja perimään (genotyyppi) kohdistuvien tyypitysmenetelmien yhdistelmää. Hippuraattitestissä käytetyn solususpension solumäärän standardointi mahdollisti hippuraattipositiivisten kantojen luotettavan tunnistuksen C. jejuni -lajiksi. Kun lajispesifisiä geenejä tunnistettiin PCR:n avulla, todettiin noin 30%:n hippuraattinegatiivisista kannoista olevan C. jejuni -lajia. Negatiivisen testituloksen perusteella ne oli kliinisen mikrobiologian laboratorioissa nimetty C. coli -lajiksi. Työssä tutkittiin kolmen tyypitysmenetelmän, serotyypityksen, PCR-RFLP-analyysin ja pulssikenttäelektroforeesin (PFGE), soveltuvuutta C. jejuni kantojen epidemiologiseen seurantaan. Serotyypitysmenetelmän erottelukykyä alensi tyypittymättömien kantojen suuri osuus. PCR-RFLP-tyypitys oli hyvin erotteleva sekä serotyypittyville että -tyypittymättömille kannoille. Serotyyppien ja RFLPtyyppien vastaavuus ei kuitenkaan ollut riittävän suuri, jotta PCR-RFLPmenetelmää voisi käyttää serotyypittymättömien kantojen molekyylitasoiseen serotyypitykseen. PFGE oli hyvin erotteleva menetelmä. KpnI-entsyymillä saavutettu erottelukyky oli lähes yhtä hyvä kuin SmaI ja KpnI -entsyymien yhteensä. Kampylobakteeri-infektioille tyypillinen vuodenaikaisjakauma, jossa tartuntamäärät ovat kesällä korkeita ja talvella matalia, johtui pääasiassa kotimaisten tartuntojen määrän vaihtelusta. C. jejuni -kannoista 41 % oli kotimaista alkuperää, C. coli -kannoista vain 17%. Serotyypit Pen 12, Pen 6,7 ja Pen 27 liittyivät tilastollisesti merkitsevästi kotimaisiin tartuntoihin, Pen 1,44, Pen 3 ja Pen 37 puolestaan ulkomaanmatkalla saatuihin tartuntoihin. Pen 2 ja Pen 4 -kompleksi olivat yleisiä serotyyppejä sekä kotimaisissa että ulkomaanmatkaan liittyvissä tartunnoissa. Serotyyppi Pen 2 oli harvinaisempi 60-vuotiailla ja sitä vanhemmilla potilailla kuin nuoremmilla ja se oli yleisempi Länsi-Suomessa kuin muualla maassa. Lisäksi se oli muita serotyyppejä yleisempi talvella. Pen 2 -tartuntojen lähde voi liittyä nautakarjaan, koska Pen 2 on yleisin serotyyppi suomalaisissa naudoissa. Kesällä 2003 potilaista eristettyjen kantojen PFGE-genotyyppejä verrattiin samaan aikaan kanoista eristettyihin kantoihin ja koko vuonna naudoista eristettyihin kantoihin. Samanlaisten tyyppien esiintymisen perusteella 31 % väestön infektioista voisi liittyä kanoihin ja 19 % nautoihin. Kolmen vuoden aikana potilaista eristetyistä kannoista ensimmäisen ja kolmannen keräysvuoden kantoja tyypitettiin PFGE-menetelmällä. Kotimaisista tartunnoista eristetyistä kannoista 69 % kuului sellaisiin SmaI-PFGE-tyyppeihin, joita esiintyi molempina vuosina. Neljä SmaI-tyyppiä kattoi 45% kotimaisista kannoista. Nämä tyypitettiin myös KpnI-entsyymillä, jolloin löydettiin kuusi molempina keräysvuosina yleistä SmaI/KpnI-tyyppiä. Nämä genotyypit esiintyivät vain satunnaisesti ulkomaisissa kannoissa, joten ne olivat pysyviä kotimaisia genotyyppejä ja liittyivät serotyyppeihin Pen 2, Pen 12, Pen 27, Pen 4 -complex, Pen 41 ja Pen 57. Samoja PFGE-tyyppejä esiintyi myös kanoissa, kahdella genotyypillä (S7/K1 ja S64/K19) oli ajallinen yhteys väestön infektioihin vuonna 2003. Viisi tyypeistä (S5/K27, S7/K1, S7/K2, S7/K5 ja S64/K19) esiintyi naudoissa. Naudat voivat erittää samaa kampylobakteerikantaa pitkäaikaisesti, joten nautakarja on todennäköisempi lähde pysyvien tyyppien aiheuttamille infektioille

Comparative Typing of Campylobacter jejuni by Heat-Stable Serotyping and PCR-Based Restriction Fragment Length Polymorphism Analysis

Campylobacter jejuni has become the most common bacterial cause of human gastroenteritis worldwide. Rapid, discriminatory typing methods are required to identify potential clusters of infections. The major disadvantage of the well-evaluated and widely used Penner heat-stable serotyping method is the high level of nontypeability. The correlation of the types determined by the Penner heat-stable serotyping method and PCR-based restriction fragment length polymorphism (RFLP) analysis of the lipooligosaccharide (LOS) biosynthesis genes of C. jejuni was studied with 149 C. jejuni strains. Of these strains, 79 were patient strains belonging to 25 Penner serotypes, 60 were nontypeable patient strains, and 10 were reference strains. A 9.6-kb DNA fragment of the LOS gene cluster was amplified and digested with the restriction enzymes HhaI and DdeI. Altogether, 39 different RFLP types (including 30 HhaI profiles and 32 DdeI profiles) were identified. Type Hh1Dd1 was the most common type, with 36% of the strains and strains of 12 serotypes being of this type. A high level of discrimination was obtained, and a correlation between the Penner serotypes and the PCR-RFLP types could be seen. Also, variation in the LOS biosynthesis genes within a single Penner serotype was found. Although the PCR-RFLP method may not be sufficient to compensate for Penner serotyping, it can give valuable information about nontypeable strains and further characterize strains of common serotypes

Chickens and Cattle as Sources of Sporadic Domestically Acquired Campylobacter jejuni Infections in Finland▿

A substantial sampling among domestic human campylobacter cases, chicken process lots, and cattle at slaughter was performed during the seasonal peak of human infections. Campylobacter jejuni isolates (n = 419) were subtyped using pulsed-field gel electrophoresis with SmaI, and isolates representing overlapping types (n = 212) were further subtyped using KpnI for restriction. The SmaI/KpnI profiles of 55.4% (97/175) of the human isolates were indistinguishable from those of the chicken or cattle isolates. The overlapping SmaI/KpnI subtypes accounted for 69.8% (30/43) and 15.9% (32/201) of the chicken and cattle isolates, respectively. The occurrence of identical SmaI/KpnI subtypes with human C. jejuni isolates was significantly associated with animal host species (P < 0.001). A temporal association of isolates from chickens and patients was possible in 31.4% (55/175) of the human infections. Besides chickens as sources of C. jejuni in the sporadic infections, the role of cattle appears notable. New approaches to restrict the occurrence of campylobacters in other farm animals may be needed in addition to hygienic measures in chicken production. However, only about half of the human infections were attributable to these sources

Antimicrobial Susceptibilities of Multidrug-Resistant Campylobacter jejuni and C. coli Strains: In Vitro Activities of 20 Antimicrobial Agents ▿

There is a paucity of information regarding antimicrobial agents that are suitable to treat severe infections caused by multidrug-resistant Campylobacter spp. Our aim was to identify agents that are potentially effective against multiresistant Campylobacter strains. The in vitro activities of 20 antimicrobial agents against 238 Campylobacter strains were analyzed by determining MICs by the agar plate dilution method or the Etest. These strains were selected from 1,808 Campylobacter isolates collected from Finnish patients between 2003 and 2005 and screened for macrolide susceptibility by using the disk diffusion test. The 238 strains consisted of 183 strains with erythromycin inhibition zone diameters of ≤23 mm and 55 strains with inhibition zone diameters of >23 mm. Of the 238 Campylobacter strains, 19 were resistant to erythromycin by MIC determinations (MIC ≥ 16 μg/ml). Given that the resistant strains were identified among the collection of 1,808 isolates, the frequency of erythromycin resistance was 1.1%. All erythromycin-resistant strains were multidrug resistant, with 18 (94.7%) of them being resistant to ciprofloxacin (MIC ≥ 4 μg/ml). The percentages of resistance to tetracycline and amoxicillin-clavulanic acid (co-amoxiclav) were 73.7% and 31.6%, respectively. All macrolide-resistant strains were susceptible to imipenem, meropenem, and tigecycline. Ten (52.6%) multiresistant strains were identified as being Campylobacter jejuni strains, and 9 (47.4%) were identified as being C. coli strains. These data demonstrate that the incidence of macrolide resistance was low but that the macrolide-resistant Campylobacter strains were uniformly multidrug resistant. In addition to the carbapenems, tigecycline was also highly effective against these multidrug-resistant Campylobacter strains in vitro. Its efficacy for the treatment of human campylobacteriosis should be evaluated in clinical trials