Clinical Isolates Of Campylobacter Jejuni Research Articles

Antibiotic resistance of Campylobacter jejuni isolates recovered from humans with diarrhoea in Turkey.

This study was aimed at investigating the occurrence and genetic mechanisms of resistance to ciprofloxacin, tetracycline and erythromycin in clinical isolates of Campylobacter jejuni recovered from human cases of acute gastroenteritis in Turkey. MIC values of each antibiotic were determined with the epsilometer test (E-test). Resistance genes/mutations were first screened by PCR and analysed by subsequent DNA sequencing. From a total of 152 C. jejuni isolates tested, 113 (74.3%), 38 (25%) and 9 (5.9%) were found to be resistant to ciprofloxacin, tetracycline and erythromycin, respectively. Sequence analysis of ciprofloxacin-resistant isolates showed that all resistant strains (n=113) carried Thr-86-Ile substition in the gyrA gene, which is the most frequently observed mutation in fluoroquinolone-resistant Campylobacter. All of the tetracycline-resistant isolates (n=38) carried the tetO gene. All of the erythromycin-resistant isolates (n=9) harboured the point mutation A2075G in the 23S rRNA gene, which is the most common mutation conferring macrolide resistance in C. jejuni. The phenotypic susceptibility testing results were found to agree well with those obtained by genetic detection methods for the C. jejuni isolates tested. The findings of this study showed a very high level of resistance to ciprofloxacin and to a lesser extent to tetracycline while resistance to erythromycin remained at a low level. Thus, erythromycin may be considered as the first choice for treatment of Campylobacter infections in this geographical region when indicated.

Antimicrobial susceptibility of clinical isolates of Campylobacter jejuni from New South Wales, Australia.

The aim of this study was to investigate the prevalence of resistance to commonly used antimicrobials in Campylobacter jejuni isolates from clinical faecal samples in New South Wales (NSW), Australia. A total of 117 C. jejuni isolates from human faecal samples from regional and metropolitan NSW were examined for antimicrobial resistance. Of the 117 isolates tested, 15.4% were resistant to ampicillin, 5.1% to tetracycline and 13.7% to ciprofloxacin. Most of the isolates were susceptible to erythromycin, except for three that showed intermediate resistance. Furthermore, 9.4% of isolates were resistant (or intermediate-resistant) to more than one antimicrobial agent. Isolates that were resistant to ampicillin and tetracycline harboured the blaOXA-61 and tet(O) genes, respectively. A mutation in the gyrA gene, resulting in the T86I substitution, was identified in the majority of ciprofloxacin-resistant isolates. The data obtained in the current study demonstrate that the majority of C. jejuni isolates evaluated were susceptible to one or more antimicrobials tested. Apart from three isolates that demonstrated intermediate resistance, all of the isolates were susceptible to erythromycin, which is the drug of choice for treating Campylobacter infections.

Reply to "Activity of Fosfomycin on Clinical Isolates of Campylobacter jejuni and Campylobacter coli of Enteric Origin".

Reply to "Activity of Fosfomycin on Clinical Isolates of Campylobacter jejuni and Campylobacter coli of Enteric Origin".

Polymerase Chain Reaction confirmation and AST test of Campylobacter jejuni from sheep of Debrebirhan Agricultural Research Center North Shoa Ethiopia

Thermophilic Campylobacter have been recognized as an important cause of food born bacteria resulting in a diarrhea in different places of the world. There are few reports on the susceptibilities of thermotolerant Campylobacter strains in human, animal and foods of animal origin in Ethiopia. During a 9 month period from August 2011 to April 2012, 43 Campylobacter jejuni were isolated from fecal and carcass swab samples respectively on blood free mCCDA agar plate in micro aerobic atmosphere and were confirmed by Polymerase Chain Reaction (PCR). The antimicrobial susceptibility test was carried out for forty three clinical isolate of Campylobacter jejuni by agar disc diffusion method. Most of the isolates tested were 100 % susceptible to penicillin and nalidixic acid 97.7% gentamicin and ciprofloxacin, 95.3 % streptomycin and chloramphenicol, 90.7% to erythromycin, 74.4 % to tetracycline and 65.1% to ampicillin. Resistance was found 34.9% to ampicillin, 23.3% to tetracycline and 7% erythromycin with only 2.3% intermediate to tetracycline and erythromycin, whereas none was susceptible isolate to cephalothin. In our study area penicillin, streptomycin, and nalidixic acid show good antimicrobial activities against Campylobacter.

Antibiotic susceptibility of clinical isolates of Campylobacter jejuni and Campylobacter coli in Ontario, Canada during 2011–2013

A total of 219 clinical isolates of Campylobacter spp. including 180 Campylobacter jejuni and 39 Campylobacter coli were assessed for in vitro antimicrobial susceptibility. Resistance among C. coli was higher for ciprofloxacin (41% versus 30.80%), erythromycin (12.80% versus 3.90%) and lower for tetracycline (53.80% versus 64.60%) compared to C. jejuni.

A SpoT polymorphism correlates with chill stress survival and is prevalent in clinical isolates of Campylobacter jejuni

Resistance of Campylobacter jejuni to environmental stress is regarded as a risk factor for the transmission of C. jejuni from poultry or poultry products to humans. So far, the mechanisms underlying the capacity of C. jejuni to survive environmental stress conditions are not fully understood. In this study, we searched for polymorphisms in C. jejuni genes, potentially involved in resistance to chill stress. To this end, we assessed 3 groups of C. jejuni isolates (clinical, retail chicken meat, and feces) for survival of experimentally induced chill stress. For each isolate we sequenced 3 genes encoding the C. jejuni sigma factors FliA, RpoD, and RpoN as well as the genes for the transcriptional regulator SpoT and the periplasmic protein HtrA. Data suggest a higher prevalence of a specific polymorphism in spoT in clinical isolates compared with poultry meat or farm isolates. Moreover, this genotype correlated with enhanced survival of chill stress. The observation that the prevalence of this SNP is relatively high in clinical isolates, which most likely have been exposed to multiple forms of stress, suggest that this SNP may be a biomarker for enhanced survival of stress.

Detection of gyrA Mutation Among Clinical Isolates of Campylobacter jejuni Isolated in Egypt by MAMA-PCR

Campylobacter spp are the major cause of enteritis in humans and more than 90% of reported infections are caused by Campylobacter jejuni. Fluoroquinolones such as ciprofloxacin are the antibiotics of choice for treatment. An increase in the frequency of ciprofloxacin-resistant Campylobacter has been reported globally due to a single base mutation (C-257 to T) in codon 86 of the quinolone resistance determining region (QRDR) of the gyrA gene altering the amino acid sequence from threonine at position 86 to isoleucine (Thr-86 to Ile). Campylobacter spp (n = 118) were selected from a collection of Egyptian isolates spanning 1998 to 2005. The presence of C. jejuni gyrA gene was confirmed in each isolate by a PCR assay amplifying 368 bp portion of the gyrA gene. C to T alteration was detected by the mismatch amplification mutation assay MAMA PCR. The MIC of nalidixic acid (NA) and ciprofloxacin (CIP) was determined by E-test. C. jejuni gyrA gene was detected in 100 of the Campylobacter spp studied; the other 18 isolates were found to be Campylobacter coli by lpxA PCR. The mutation was detected in 89 C. jejuni resistant isolates with MIC values (NA; 8 - >256 μg/ml) and (CIP; 4 - >32 μg/ml). The other 11 sensitive C. jejuni isolates with MIC values (NA; 0.38 - 3 µg/ml) and (CIP; 0.03 - 0.125 µg/ml) were not amplified by the MAMA primers. There was 100% congruence with MAMA PCR, MIC results and gyrA gene sequence analysis. In Egypt the main mechanism for resistance to fluoroquinolones is an alteration in the gyrA QRDR. MAMA PCR provides an economical and rapid means for screening fluoroquinolone resistance.

Transposon mutagenesis in a hyper-invasive clinical isolate of Campylobacter jejuni reveals a number of genes with potential roles in invasion

Transposon mutagenesis has been applied to a hyper-invasive clinical isolate of Campylobacter jejuni, 01/51. A random transposon mutant library was screened in an in vitro assay of invasion and 26 mutants with a significant reduction in invasion were identified. Given that the invasion potential of C. jejuni is relatively poor compared to other enteric pathogens, the use of a hyper-invasive strain was advantageous as it greatly facilitated the identification of mutants with reduced invasion. The location of the transposon insertion in 23 of these mutants has been determined; all but three of the insertions are in genes also present in the genome-sequenced strain NCTC 11168. Eight of the mutants contain transposon insertions in one region of the genome (approximately 14 kb), which when compared with the genome of NCTC 11168 overlaps with one of the previously reported plasticity regions and is likely to be involved in genomic variation between strains. Further characterization of one of the mutants within this region has identified a gene that might be involved in adhesion to host cells.

Role of the plasmid-encoded tet(O) gene in tetracycline-resistant clinical isolates of Campylobacter jejuni and Campylobacter coli

The prevalence of tetracycline resistance, tetracycline MICs and tet(O) gene localization were investigated in 83 Campylobacter isolates from patients suffering from acute gastroenteritis in Germany. Combined biochemical and molecular markers identified 74 isolates (89 %) as Campylobacter jejuni, including seven atypical isolates that failed to hydrolyse hippurate, and nine isolates (11 %) as Campylobacter coli. Tetracycline resistance was detected in six out of nine Campylobacter coli isolates (67 %) and 13 out of 74 C. jejuni isolates (18 %). Low-level tetracycline resistance was observed for C. coli (MIC 16 microg ml(-1) for all strains), whereas C. jejuni showed high-level resistance (MIC >256 microg ml(-1) for all strains). Both low- and high-level tetracycline resistance was associated with the presence of the tet(O) gene. In C. jejuni, tet(O) was plasmid-encoded in 54 % of tetracycline-resistant isolates, whereas in C. coli, tet(O) appeared to be located on the chromosome.

Identification of putative zinc hydrolase genes of the metallo-β-lactamase superfamily fromCampylobacter jejuni

DNA fragments encoding two putative zinc-dependent hydrolases, designated GLX2-1 and GLX2-2, from a clinical isolate of Campylobacter jejuni, strain 012, were cloned and sequenced. GLX2-1 was encoded by a sequence of 798 bp and GLX2-2 by a sequence of 597 bp. The amino acid sequences deduced from C. jejuni DNA showed 99% and 100% identity, respectively, to putative zinc hydrolases reported from C. jejuni ATCC strain 11168, and also shared identity (28-43%) with several hypothetical conserved proteins and known zinc-dependent hydrolases and metallo-beta-lactamase superfamily proteins. A strictly conserved motif, -H-X-H-X-D-, characteristic of the metallo-beta-lactamase superfamily of proteins, including class B metallo-beta-lactamases, was identified in both proteins. Other conserved metal-binding ligands, characteristic of the metallo-beta-lactamase superfamily of proteins, were also identified. Functional beta-lactamase could not be expressed in either Escherichia coli or Campylobacter coli transformed with C. jejuni hydrolase-containing plasmids, suggesting that they do not function as metallo-beta-lactamases, although structurally they are consistent with the zinc metallo-hydrolase family of the beta-lactamase fold.

Effect of oral administration of Butyrivibrio fibrisolvens MDT-1 on experimental enterocolitis in mice.

Butyrivibrio fibrisolvens MDT-1, a butyrate-producing strain, was evaluated for use as a probiotic to prevent enterocolitis. Oral administration of the MDT-1 strain (10(9) CFU/dose) alleviated the symptoms of colitis (including body weight loss, diarrhea, bloody stool, organic disorder, and mucosal damage) that are induced in mice drinking water that contains 3.0% dextran sulfate sodium. In addition, myeloperoxidase (MPO) activity levels in colonic tissue were reduced, suggesting that MDT-1 mitigates bowel inflammation. The addition of MDT-1 culture supernatant inhibited the growth of nine clinical isolates of Campylobacter jejuni and Campylobacter coli that could potentially cause enterocolitis. Infection of mice with C. coli 11580-3, one of the isolates inhibited by MDT-1 in vitro, resulted in diarrhea, mucosal damage, increased MPO activity levels in colonic tissue, increased numbers of C. coli in the cecum, and decreased body weight gain. However, administration of MDT-1 to mice, prior to and during C. coli infection, reduced these effects. These results suggest that Campylobacter-induced enterocolitis can be alleviated by using B. fibrisolvens as a probiotic.

Sequence analysis of a cryptic plasmid pCJ419 from Campylobacter jejuni and construction of an Escherichia coli– Campylobacter shuttle vector

A small cryptic plasmid, pCJ419, was identified in a human clinical isolate of Campylobacter jejuni, cloned and sequenced. pCJ419 is a circular molecule of 4013 bp with a G + C content of 27.1%. The products of four open reading frames (ORFs) share significant sequence similarity with putative proteins from known C. jejuni and Campylobacter coli plasmids. ORF-1 encodes a putative mobilisation protein (Mob). ORF-2 and ORF-3 encode proteins that have high identity to putative RepA and RepB proteins, respectively, of known C. jejuni and C. coli plasmids. ORF-4 encodes a protein that has high identity to a hypothetical protein of unknown function, Cjp32, previously described in a pVir plasmid of C. jejuni. Tandem repeating 22-bp sequences typical of a plasmid replication origin ( ori) were identified upstream of the DNA sequences encoding putative replication initiation proteins. An Escherichia coli– Campylobacter shuttle cloning vector, pGU0202, was constructed using plasmid pMW2 that harbours a Campylobacter-derived kanamycin resistance gene [ aph(3 ′)- III]. The sequences encoding pCJ419 mob, RepA and RepB proteins were inserted upstream of aph(3 ′)- III resulting in a stable construct of 6174 bp that was used to transform both E. coli and Campylobacter.

The tetracycline resistance protein Tet(o) perturbs the conformation of the ribosomal decoding centre.

Tet(o) is an elongation factor-like protein found in clinical isolates of Campylobacter jejuni that confers resistance to the protein-synthesis inhibitor tetracycline. Tet(o) interacts with the 70S ribosome and promotes the release of bound tetracycline, however, as shown here, it does not form the same functional interaction with the 30S subunit. Chemical probing demonstrates that Tet(o) changes the reactivity of the 16S rRNA to dimethyl sulphate (DMS). These changes cluster within the decoding site, where C1214 is protected and A1408 is enhanced to DMS reactivity. C1214 is close to, but does not overlap, the primary tetracycline-binding site, whereas A1408 is in a region distinct from the Tet(o) binding site visualized by cryo-EM, indicating that Tet(o) induces long-range rearrangements that may mediate tetracycline resistance. Tetracycline enhances C1054 to DMS modification but this enhancement is inhibited in the presence of Tet(o) unlike the tetracycline-dependent protection of A892 which is unaffected by Tet(o). C1054 is part of the primary binding site of tetracycline and A892 is part of the secondary binding site. Therefore, the results for the first time demonstrate that the primary tetracycline binding site is correlated with tetracycline's inhibitory effect on protein synthesis.

Differentiation of Campylobacter species by AFLP fingerprinting

The fluorescent amplified fragment length polymorphism (AFLP) fingerprinting method was tested for its ability to identify and subtype the most important Campylobacter species found in veterinary infections. Sixty-nine reference strains and 19 clinical isolates of Campylobacter jejuni subsp. jejuni, Campylobacter jejuni subsp. doylei, Campylobacter upsaliensis, Campylobacter coli, Campylobacter lari, Campylobacter fetus subsp. fetus, C. fetus subsp. venerealis, Campylobacter hyointestinalis subsp. hyointestinalis, C. hyointestinalis subsp. lawsonii, Campylobacter mucosalis, Campylobacter helveticus and Campylobacter sputorum were subjected to analysis. The topology of the dendrogram obtained by numerical analysis of the AFLP profiles did not reflect the phylogenetic relationships as derived from 16S rDNA sequence comparison. However, except for C. lari, AFLP analysis grouped the strains that belonged to the same genomic species into distinct clusters. C. lari strains were separated into two distinct AFLP groups, which corresponded with nalidixic-acid-sensitive and -resistant variants of C. lari. These results correlated with data from whole-cell protein profiling. Within C. jejuni, C. hyointestinalis and C. fetus, strains could be identified at the subspecies level. AFLP analysis also allowed the subtyping of most species at the strain level. It is concluded that AFLP analysis is a valuable tool for concurrent identification of campylobacters at the species, subspecies and strain levels. In addition, the data confirm and extend previous reports showing that C. lari is a heterogeneous species that may comprise multiple taxa.

Single or double mutational alterations of gyrA associated with fluoroquinolone resistance in Campylobacter jejuni and Campylobacter coli.

We looked for the presence of gyrA mutations in seven fluoroquinolone-resistant French clinical isolates of Campylobacter jejuni and Campylobacter coli. Three of the five isolates of C. jejuni and the two isolates of C. coli had high-level resistance to nalidixic acid (MICs 128-256 microg/ml) and ciprofloxacin (MICs 32 microg/ml). A gyrA mutation was found in all these isolates leading to the following substitutions: Thr86-Ile in four cases and Asp90-Tyr for one C. coli strain. One isolate had high-level resistance to nalidixic acid (MIC 64 microg/ml) but low-level resistance to ciprofloxacin (MIC 2 microg/ml) and also carried a gyrA mutation leading to a Thr86-Ala substitution. The last isolate of C. jejuni studied displayed an atypical resistance phenotype: It was resistant to high levels of ciprofloxacin (MIC 64 microg/ml) but remained fully susceptible to nalidixic acid (MIC 2 microg/ml). This phenotype was not explained by the presence of peculiar mutations in gyrA or gyrB. It carried a gyrA mutation leading to a Thr86-Ile substitution and was devoid of gyrB mutation. Despite numerous attempts with various degenerate oligonucleotide primers deduced from conserved regions of known parC genes, we were unable to amplify a corresponding sequence in C. jejuni or C. coli. First-step and second-step in vitro mutants, derived from reference strain C. coli ATCC 33559 with ciprofloxacin or moxifloxacin as selecting agents, were found to carry one and two mutations in gyrA, respectively. In contrast with the results obtained with clinical isolates, a variety of gyrA mutations were obtained in vitro.

An integron cassette carrying dfr1 with 90-bp repeat sequences located on the chromosome of trimethoprim-resistant isolates of Campylobacter jejuni.

The frequent occurrence of high-level trimethoprim resistance in clinical isolates of Campylobacter jejuni was shown to be related to the acquisition of foreign resistance genes (dfrl or dfr9 or both) coding for resistant variants of the enzyme dihydrofolate reductase, the target of trimethoprim. The dfr1 gene detected on the chromosome of 40 different clinical strains of C. jejuni was studied further regarding structure and genetic organization. Most of the dfr1 genes were found as integron cassettes inserted in the chromosome. In 36% of the examined isolated, the dfr1 gene showed identity to that previously characterized in trimethoprim-resistant Escherichia coli. In 40% of the cases, however, a variant of the dfr1 gene containing a 90-bp direct repeat was detected, and in 5% of the isolates, the repeat-containing dfr1 variant was found to occur in the form of two cassettes in tandem in an integron context. The existence of the 90-bp repeat within the coding sequence of the dfr1gene was found to play a role in the adaptation of C. jejuni to ambient concentrations of trimethoprim.

Identification and characterisation of a cytotoxic porin-lipopolysaccharide complex from Campylobacter jejuni.

A clinical isolate of Campylobacter jejuni, previously found to produce a toxin active in cell culture assays, was used for identification and characterisation of a cytotoxic porin-lipopolysaccharide (LPS) complex. This cytotoxic complex was isolated by high-performance liquid chromatography of crude concentrated culture supernate and DEAE-anion exchange chromatography. The complex had a toxic activity of 20.1 tissue culture dose50 (TCD50)/microg of protein for HEp-2 cells, 7.49 TCD50/microg of protein for HeLa cells and 1.87 TCD50/microg of protein for Chinese hamster ovary cells. Analysis by SDS-PAGE revealed a single protein band of 45 kDa and a high mol. wt carbohydrate moiety. The complex gave a positive result in the Limulus amoebocyte lysate test, indicating that the co-purifying carbohydrate was LPS, and had specificity for the lectins Galanthus nivalis agglutinin, Maackia amurensis agglutinin and Datura stramonium agglutinin. The cytotoxic activity associated with the complex was heat-labile at 70 degrees C, resistant to inactivation with trypsin and retained activity after treatment with sodium metaperiodate and the glycosidases neuraminidase and N-glycosidase F. Sequencing of the N-terminus of the protein component of the complex revealed 97% homology with the major outer-membrane porin protein from C. jejuni. The cytotoxic activity of the complex was neutralised by a polyclonal, homologous antiserum, which reacted on Western blot with the 45-kDa protein, but not by polyclonal antisera raised against a number of other bacterial toxins.

High-level resistance to trimethoprim in clinical isolates of Campylobacter jejuni by acquisition of foreign genes (dfr1 and dfr9) expressing drug-insensitive dihydrofolate reductases.

The pathogenic bacterium Campylobacter jejuni has been regarded as endogenously resistant to trimethoprim. The genetic basis of this resistance was characterized in two collections of clinical isolates of C. jejuni obtained from two different parts of Sweden. The majority of these isolates were found to carry foreign dfr genes coding for resistant variants of the dihydrofolate reductase enzyme, the target of trimethoprim. The resistance genes, found on the chromosome, were dfr1 and dfr9. In about 10% of the strains, the dfr1 and dfr9 genes occurred simultaneously. About 10% of the examined isolates were found to be negative for these dfr genes and showed a markedly lower trimethoprim resistance level than the other isolates. The dfr9 and dfr1 genes were located in the context of remnants of a transposon and an integron, respectively. Two different surroundings for the dfr9 gene were characterized. One was identical to the right-hand end of the transposon Tn5393, and in the other, the dfr9 gene was flanked by only a few nucleotides of a Tn5393 sequence. The insertion of the dfr9 gene into the C. jejuni chromosome could have been mediated by Tn5393. The frequent occurrence of high-level trimethoprim resistance in clinical isolates of C. jejuni could be related to the heavy exposure of food animals to antibacterial drugs, which could lead to the acquisition of foreign resistance genes in naturally transformable strains of C. jejuni.

Evolution of susceptibilities of Campylobacter jejuni isolated from diarrhoeal cases to fluoroquinolones in Tokyo

Recently, the increase in the number of resistant strains of Campylobacter jejuni to fluoroquinolone has been reported in European countries. We also studied antimicrobial susceptibilities of 600 clinical isolates of Campylobacter jejuni isolated during a 6 year period from 1989 through 1994 in four Tokyo Metropolitan Hospitals. The susceptibility to 6 antimicrobial agents, norfloxacin (NFLX), ofloxacin (OFLX), ciprofloxacin (CPFX), nalidixic acid (NA), erythromycin (EM) and tetracycline (TC) were examined. The overall resistant rates were as follows: NFLX, 45 strains (7.5%); OFLX, 45 strains (7.5%); CPFX, 44 strains (7.3%); NA, 62 strains (10.3%); EM, 4 strains (0.6%) and TC, 259 strains (43.2%). The number of resistant strains to fluoroquinolones and NA has increased significantly since 1993 in Japan, but the susceptibility to erythromycin has still remained the same level during the past 6 years. The susceptibility to TC was variable, and MICs gave a bimobal distribution, as pointed out previously. The resistance pattern of NFLX, OFLX, CPFX and NA were observed most frequently in those isolates.

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The Lior serotype reference strains and clinical isolates of Campylobacter jejuni were compared in the restriction fragment length polymorphism (RFLP) pattern to distinguish C. jejuni strains. These reference strains showed RFLP patterns different from one to another, while the patterns of some isolates were not coincident with those of the same serotype reference strains. Furthermore, we tried to hybridize HindIII-digested fragments from these strains with the DNA probe encoding the 46-kDa protein of C. jejuni by Southern and slot blottings. The 1.8-kbp fragments from all strains of C. jejuni hybridized with this probe, but those from other species of Campylobacter or enterobacteria did not. These results indicate that the Lior serotype is unrelated with the RFLP pattern of DNA of C. jejuni strains, but the DNA probe is useful to detect C. jejuni.