African Journal of
Microbiology Research

  • Abbreviation: Afr. J. Microbiol. Res.
  • Language: English
  • ISSN: 1996-0808
  • DOI: 10.5897/AJMR
  • Start Year: 2007
  • Published Articles: 5177

Full Length Research Paper

Use of a combined cultural-molecular method for isolation and identification of Campylobacter from broiler chicken in Morocco

Charrat Nadia*
  • Charrat Nadia*
  • Laboratory of Microbiology and Molecular Biology, Department of Food Microbiology, Research Laboratory and Medical Analysis (LRAM), Fraternal of the Royal Gendarmerie, Rabat, Morocco.
  • Google Scholar
El Fahime Elmostafa
  • El Fahime Elmostafa
  • Laboratory of Sequencing and Genotyping, National Center for the Scientific and Technical Research (CNRST), Rabat, Morocco.
  • Google Scholar
Filali-Maltouf Abdelkarim
  • Filali-Maltouf Abdelkarim
  • Laboratory of Microbiology and Molecular Biology, Faculty of Science (FSR), University Mohammed V, Rabat, Morocco.
  • Google Scholar


  •  Received: 23 November 2016
  •  Accepted: 31 January 2017
  •  Published: 21 February 2017

 ABSTRACT

Campylobacters are very important foodborne pathogens that raise the interest of food processors, researchers, as well as consumers and all stakeholders. Their contaminations can result in life threatening disorders, potentially leading to chronic sequelae such as Reiter’s and Guillain-Barré syndromes or Crohn’s Disease. Poultry has been identified as the main and most common reservoirs for Campylobacter. A survey was conducted in Morocco, from 2009 to 2012 to estimate the prevalence of Campylobacter in broiler chicken from about fifteen Moroccan cities that were most involved in the breeding of broilers. In this study, thermotolerant Campylobacter spp. were detected and identified from 165 samples by both cultural methods and molecular approaches based on polymerase chain reaction  and 16S ribosomal RNA (rRNA) gene sequencing. The species were also genotyped by Restriction Fragment Length Polymorphism analysis. The conventional culture methods identified 97% of samples as positive for Campylobacter spp. The molecular approach based on 16S rRNA gene could not distinguish between C. jejuni, C. coli and C. lari. However, gyrB gene RFLP, allowed a good discrimination between the three species of Campylobacter. These results were also confirmed by Matrix Assisted Laser Desorption Ionization-Time of Flight and Mass Spectrometry proteomic profiling determination using libraries in the BioTyper 2.3 software. The present study identifies C. jejuni as the major source of contamination of poultry carcasses in Morocco.

Key words: Campylobacter, chicken, 16S rRNA gene, gyrB gene, sequencing, RFLP-PCR, MALDI-TOF MS.


 INTRODUCTION

Campylobacteriosis is a zoonosis, renowned as the  most Frequently listed food borne illness in humans at  international level. The high incidence of campylobacteriosis as well as its duration and the possible after-effects make it highly important from a socio-economic perspective. The World Health Organization considers Campylobacter like the most common bacterial cause of human gastroenteritis in the world. (WHO, 2016). Campylobacter causes more cases of diarrhea compared to other foodborne pathogens like Salmonella or Yersinia, both in developing and developed countries (Humphries and Schuetz, 2015). In developing countries, Campylobacter infections among children under the age of two years as well as elderly and immunosuppressed individuals are particularly frequent, sometimes result in death (Samosornsuk et al., 2015). Campylobacter is reported as a causative agent of diarrhea among travellers staying repeatedly in developing areas including the Middle East, North Africa, Southeast Asia and Latin America (Kittitrakul et al., 2015). About seventeen (17) species, six (6) subspecies and four (4) biovars have been assigned to the Campylobacter genus, however, the most infectious species that has been frequently reported in humans are C. jejuni (subspecies jejuni) and, to a lesser extent, C. coli, and C. lari. Other species such as C. hyointestinalis, C. upsaliensis, and C. sputorum have also been isolated from patients with diarrheal disease, but are reported to be less frequent (Boelaert, 2016).
 
Campylobacter infection in humans may be due to the several reasons which include consumption or contact with undercooked poultry meat (Umaraw et al., 2017), cross-contamination from raw poultry, meat, foods that are in poor conservation conditions (potentially at low doses) or those which are commonly eaten without further heating (MacDonald et al., 2015). Campylobacters are readily detectable in the faeces of colonized birds, especially in chicken (Ishihara et al., 2016). In 2011, European Food Safety Authority (EFSA) has found that chickens and chicken meat might directly account for 20-30% of human cases (EFSA, 2011; Di Giannatale et al., 2016). As reported in the “Review of the poultry sector” of the FAO/UNO (Food and Agriculture Organization/United Nations Organization), in Morocco, the poultry sector constitutes one of the most dynamic agricultural fields where traditional poultry sector still provides local markets with white meats (13.5%) and broiler chicken is the most appreciated because it is an accessible source of meat to all social classes (Barkok, 2008).
 
Since its starting, at the beginning of the seventies, the intensive Moroccan avicolous sector developed in the absence of lawful measurements specific to this activity. However, since the nineties, the poultry industry has undergone a major expansion with the installation of a large number of livestock farms. The number of broiler farms has more than 5,000 units and many of them are located in the axis “El Jadida - Casablanca – Kénitra” due to the temperate climate of these cities,  the  proximity  of the supply centers and marketing markets. Indeed, this axis covers 42% of broiler flocks on a national level. Practically all commercial breeding units of chicken in Morocco belong to the sector 2 of the FAO’s classification. The semi- intensive farming or amateur (similar to the sector 3) are very rare. The marketing and the distribution of chicken in Morocco are done according to a primitive system; more than 90% of produced chickens are sold alive by small retailers. Those retailers supply either to markets or directly to farms. The consumer thus buys a live animal, and generally gives it then to any adjacent slaughter slaps for the service of sticking, plucking and evisceration. In Morocco, five commercial strains of exotic breeding chickens, called type “flesh”, has been valued industrially, because of their rapid growth with an average of 2 ± 0.2 kg at an age of 40 ± 5 days: Ross, Hubbard, JV (“Vedette”), Cobb and Arbor Acres. Ross, Hubbard and JV strains hold 94 % of the national market. Ross would be in the lead because of its growth rate and its consumption index. Very few hatcheries specialize in breeding a single strain. Currently, the breeding of reproducers are very organized (Barkok, 2008).
 
The prevalence of Campylobacter infections, the species distribution in bacterial enteric infections and their antimicrobial resistance patterns of Morocco still remain unclear. This makes the detection of Campylobacter crucial in preventing its spread as well as in treating the disease or establishing the epidemiology of Campylobacter infection in Morocco.
 
The purpose of this work was to make a preliminary study of prevalence of Campylobacter in our country and to compare the accuracy of the classical and Molecular sub-typing methods for species identification among isolated Campylobacter genus.


 MATERIALS AND METHODS

Ethics statement
 
The animals were kept and used in harmony with the instructions (policy, procedures and standards) of the Ethic Committee of the LRAM (Laboratoire de Recherche et d’Analyses Médicales de la Fraternelle de la Gendarmerie Royale – Morocco) which is responsible for monitoring internal researches involving the use of animals.
 
Sample collection
 
A 3-year study was performed from October 2009 to April 2012. One hundred and sixty five living broiler chickens were collected directly from commercial farms, retailers and markets of seventeen different sites.
 
We targeted fifteen Moroccan cities, which were mostly involved hatching using artificial incubators and the rearing of chickens for meat according to the review of the poultry sector of the FAO/UNO (Barkok, 2008). Namely: Rabat : from P1 at P30, Chichaoua: P31- P40, Settat: P41- P50, Tanger P51- P55, Temara: P56-P65, Nador: P66-P75, Agadir: P76-P85, Marrakech: P86-P95, El Jadida: P96-P105, Meknès: P106-P115 , Kénitra: P116-P125, Casablanca: P126-P135, Safi: P136-P145, Sefrou:  P146-P155 and Kelâa Sraghna : P156-P165. The sampling sites of living chickens in different geographical locations of Morocco are indicated in Figure 1.
 
 
For our research works, we used five (5) strains of commercial reproductive broiler chickens such as Ross, Hubbard, JV (“Vedette”), Cobb and Arbor Acres without any distinction of strains or sex.
 
Chickens were chosen with an average body weight fixed at 2 ± 0.2 kg, aged about 8 weeks (40 ± 5 days). Most studies have shown that Campylobacter contaminates flocks of chicken between the second and the fourth week of breeding (Cean et al., 2015; Gonsalves et al., 2016; Rodgers et al., 2016) this has to be included in review.
 
The broiler chickens were transported separately in plastic boxes to the laboratory animal unit of the LRAM where the euthanasia of the broilers was carried out according to national laws and rules and internal policies, respecting the animal and preventing any cross-contamination.
 
The carcasses were scalded individually, with hot water, in the traditional way to reproduce the household gestures and to facilitate the process of plucking. To avoid the thermal shocks (where Campylobacter is very sensitive), carcasses were immediately directed to the laboratory for dissection and analyses. The carcasses were eviscerated and washed thoroughly with tap and warm water. The viscera (including intestines with their faeces) were removed aseptically and also the gallbladder to escape antibacterial activity of the bile (Begley et al., 2005; Merritt and Donaldson, 2009).
 
A total of 165 viscera samples from broilers were collected and placed into individual sterile plastic bags (Whirl-Pak Sample Bags, Nasco, B01323WA; VWR International), well-identified in a sterile bag, denoted P followed by a number, then they were weighed,  mixed and homogenized thoroughly at  10,000 r.p.m.  for  30 s  in  a food crusher (Knife Mill Grindomix GM 200; Retsch).
 
 
Enrichment using selective liquid medium
 
The incorporation of enrichment procedures was to increase recovery of Campylobacter from our samples. All samples were serially diluted as per the standards (ISO 6887-1:1999), 25 g of viscera mix was weighed twice to get two subsamples in individual sterile stomacher bags (80015, bioMérieux). Each subsample was enriched in 225 ml of Preston Broth (PB) containing 5% (v/v) with Sheep Blood (Base Agar CM0689, supplement SR0204E; Oxoid) and incubated at 42°C for 18 h in a microaerophilic atmosphere using atmospheric generators and jars of incubation (GENbox Microaer, bioMérieux, France) with a microaerophilic gas mixture according to the ISO 10272 procedure, 1995.
 
Culture using selective agar
 
The detection of thermotolerant Campylobacter spp. (C. coli, C. jejuni, C. helveticus, C. lari, C. sputorum, and C. upsaliensis) by conventional bacterial culture method was conducted in accordance with the recommendations from the International Organization for Standardization (Standard Method ISO 10272 Procedures, 1995; 1996 et al., 1997).
 
Bacteria isolation
 
After incubation and for each subsample enriched PB, an aliquot of growth from an enrichment culture (initial suspension in tubes) was streaked onto Karmali Agar (Base Agar CM0935, supplement SR0205E; Oxoid) and modified Bützler Agar (Base Agar CM0271, supplement SR0085E; Oxoid). Following inoculation of sub-culture, all plates were incubated under microaerophilic condition using anaerobic jars gassed (GENbox Microaer, bioMérieux, France) at 42°C for 2 to 3 days depending on bacterial growth. After opening the jar and examining the cultures, if a second incubation was necessary, then new generators were employed.
 
Confirmation of thermotolerant Campylobacter spp
 
The identities of bacterial strains obtained from different samples were verified by microscopy and Gram staining. Standard phenotypic tests were used to identify species specific of field strains according to a previously described method (Wimalarathna et al., 2013). Strains were initially analyzed for the following properties: Gram negativity, spiral morphology, and microaerophilic growth dependency. Assays for oxidase and catalase activity as well as Hippurate hydrolysis were performed. Antimicrobial susceptibility tests with Nalidixic Acid and Cephalotin, were checked as described in ISO 10272:1995. Subsequently, Hydrogen Sulphide (H2S) production was checked in TSI medium.
 
Bacterial strains for positive control
 
In this study, bacterial strains of some Campylobacter species were used as positive control in conventional PCR assays, as well as in quality control of productivity testing of media homemade:
 
i) Campylobacter jejuni subsp jejuni (ATCC 49943; AWEEL).
ii) Campylobacter coli (ATCC 43478; Microbiologics).
iii) Campylobacter lari (CIP 102722; kindly provided by Bacteriology Service of the French National Reference Center for Campylobacter and Helicobacter).
 
Molecular assays
 
DNA extraction
 
Bacterial DNA was extracted from colonies of 150 samples presumptive of Campylobacter spp. using the Sigma’s GenElute Bacterial Genomic DNA Kit (Sigma-Aldrich, USA), according to manufacturer’s instructions. Briefly, 1.5 mL of bacterial broth culture was pelleted at 12,000 to 16,000 × g for 2 min; cells were resuspended in 180 μL lysis solution A for Gram-negative bacteria or in 200 µL of lysozyme (200 units/ml) for Gram-positive bacteria. Then, 20 μL of Proteinase K was added to the cell suspension. After incubation at 55°C for 30 min, 200 μL of lysis solution C was added to the suspension. The suspended cells were then incubated at 55°C for 10 min. DNA was purified using GenElute Miniprep Binding Columns (Sigma-Aldrich, USA). DNA eluted out in sterile distilled water and stored at -20°C until use.
 
16S rRNA gene sequencing
 
The 16S rRNA gene sequence of 52 samples taken at random from 150 samples presumptive Campylobacter spp. was amplified using primers fD1 (5’-AGA GTT TGA TCC TGG CTC AG-3′) and rP2 (5′-AAG GAG GTG ATC CAG CC-3′), as described by Weisburg et  al. (1991). PCR was carried out using 2.5 µl of 10X buffer, 1.5 mM MgCl2, 0.2 mM of each dNTPs, 0.4 µM of each primer, 1 U of Platinum Taq Polymerase (Invitrogen) and 5 µl (30 ng/µl) of template DNA in a 25 µl reaction volume under the following conditions : 4 min at 96°C (initial denaturation), 35 cycles of 10 s at 96°C (denaturation), 40 s at 52°C (annealing), 2 min at 72°C (extension), and one final step of 4 min at 72°C (extension cycle) employing the PCR thermocycler “Verity” (Applied Biosystems, Foster City). The amplified fragments were electrophoresed on 1% agarose gels and detected using ethidium bromide along with molecular weight markers. The PCR products were purified using EXOSAP-IT (USB, USA) and bidirectionally sequenced on an ABI 3130 Xl automated sequencer (Applied Biosystems, Foster City) using BigDye Terminator version 3.1 Kits using the same primers as of PCR. The analysis of electrophorogram was performed with the sequencing Analysis Software version 5.3.1 (Applied Biosystems, Foster City). The consensus sequences were edited and compared with published sequences available in GenBank, using BLAST tool of the NCBI.
 
RFLP analysis of gyrase B gene
 
For the gyrB gene amplification we used the universal primer mix described by Kawasaki et al. (Kawasaki et al., 2008) to amplify a 960-bp gyrB gene fragment from each Campylobacter reference strain and from each sample. A total of 150 samples were analysed by RFLP (Restriction Fragment Length Polymorphism).
 
The DNA template (1 µl) was amplified in a 50 µl reaction volume containing 1x PCR buffer, 2 mM MgCl2, 0.25 U rTaq, (Sigma-Aldrich, USA) DNA polymerase, a 0.4 mM concentration of each of the four (4) dNTPs, and the universal primer mixture consisting of a 10 nM concentration of each primer. The cycling conditions consisted of an initial denaturation at 94°C for 5 min, followed by 45 cycles of denaturation (94°C for 30 s), annealing (55°C for 45 s), and extension (72°C for 30 s), with a final 7-min extension at 72°C. Computational prediction of the restriction fragment length analysis
 
of the 960-bp amplified region predicted that the DdeI enzymes would generate species-specific digestion patterns (http://www.restrictionmapper.org). For RFLP analysis, the purified PCR products were digested in a total volume of 20 µl with either 5 U of DdeI (Toyobo, Osaka, Japan). The resulting fragments were size separated using 2.5% Agarose prepared in 1x Tris-acetate-EDTA buffer and stained with Sybr green I dye (Invitrogen, Carlsbad, CA). The PCR-RFLP approach was validated by comparison of the DdeI I digestion pattern of the  standard strains C. jejuni, ATCC 49943; C. coli, ATCC 43478 (Manassas, VA, USA) and C. lari, from Pasteur Institute Collection (CIP 102722) to the computational prediction of the amplicon digestion using the same restriction enzyme as shown in Figure 2. All References Campylobacter species showed species-specific DdeI digestion patterns.
 
 
Mass spectrometry microbial identification system
 
The 155 strains isolated at LRAM were analyzed in situ. We used overnight bacterial cultures after their activation following Biomérieux’s recommendations on incubation temperatures. For rapid identification of Campylobacter species, we worked on the IVD MALDI BioTyper system based on proteomic profiling and identification of bacteria directly from positive blood cultures samples using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).
 
Preparations of bacterial isolates for MALDI-TOF MS measurement were carried out as previously described (De Carolis et al., 2014; Rahi et al., 2016). Peptidic spectra were compared with the Bruker MALDI Biotyper library (version 5627) and software (version 2.3). Two distinct criteria were used to analyse the results of the spectral database search, a score value and a consistency category and Log score values range from 0.000 to 3.000 and are correlated with an explanation of genus and species consistency within the database. The software as highly probable species-level identification interprets a score ranging from 2.3 to 3.000. Log scores of between 2.00 and 2.299 represent secure genus identification and probable species-level identification.


 RESULTS

In this study, thermotolerant Campylobacter strains isolated from different geographical areas of Morocco were identified with conventional methods, 16S rRNA gene sequencing, gyrB RFLP-PCR and MALDI-TOF MS determination methods.
 
Conventional method for the isolation and characterization of Campylobacter spp.
 
The conventional bacterial identification tests including Gram staining, culture and growth characteristics and biochemical patterns showed that 160 out of the 165 samples (97%) were contaminated with thermotolerant Campylobacter strains whereas five (3%) samples were negative for Campylobacter spp. (samples: P11; P59;
P67; P79; P149).
 
The bacterial identification of the 160 strains isolated from the samples revealed that 2 samples were positive in Hippurate hydrolysis test and sensitive to nalidixic acid and were identified as C. jejuni. About twenty four (24) samples
 
were negative in Hippurate hydrolysis test and resistant to antibiotics which were recognized as C. lari. The remaining 134 samples identified as Campylobacter genus were unspecified because of their bacterial behaviour under conditions of Hippurate hydrolysis and antibiotics sensitivity reaction. These results are summarized in Table 1.
 
 
Detection and identification of Campylobacter species by molecular biology method (16S rRNA sequencing, PCR-RFLP of gyrB gene)
 
Out of the 165 samples, only 150 samples were analysed by molecular approaches because samples P1 to P10 were unable to be preserved and five samples were negative for Campylobacter.
 
16S rRNA gene sequencing
 
In this study the 16S rRNA gene were amplified for 105 samples from among the 150 samples. Sequencing of the 1540 bp 16S rRNA PCR fragments generated around  1200 to 1400-bp of sequence with an average Phred score of ≥ 20 (≥ 99% accurate). Sequences derived from the 16S rRNA genes of 105 presumptive Campylobacter strains were queried against NCBI’s bacterial sequence databases using the web-based analysis tool. The 16S rRNA sequences queries returned a list of matching sequences that were a mixture of C. jejuni, C. coli and C. lari sequences, all possessing identity scores of 100-98%. This inability to differentiate between these three species using the commonly employed 16S rRNA sequencing procedure has been previously reported (Loong et al., 2016; Vondrakova et al., 2014) which prompted  us  to  look  for  alternative
methods.
 
PCR-RFLP of gyrB gene
 
Restriction enzyme analysis of PCR amplicons, known as PCR-RFLP, is a useful tool for molecular characterization of food-borne pathogens, including differentiation of thermophilic Campylobacters (Kamei et al., 2014). We have first validated this approach by using DNA from known Campylobacter reference strain. The results showed clearly that PCR-RFLP profiles matched perfectly with the computational prediction of the in sillico-digestion of the gyrB gene sequences of each reference strain.
 
Following the validation step, the approach was also applied to the samples. The results showed that out of the 150 strains positive for Campylobacter genus, 88 (59%) were identified as C. jejuni, 40 (27%) as C. coli, 2 (1%) contained both species C. jejuni / C. coli. A case of probable coexistence of two species in the same sample is shown for (P72 and P96) (Figure3).None were recognized as C. lari and the rest (20 samples): were unspecified because of unamplified DNA.
 
 
Identification of Campylobacter species by MALDI-TOF MS
 
Compared to the standard approach, the Matrix Associated Laser Desorption Ionization–Time Of Flight Mass Spectrometry (MALDI-TOF MS) seems to be interesting in terms of rapidity and cost (Branda et al., 2014). To assess the chosen methods and confirm the obtained results (by culture and molecular biology), we tested the 150 strains isolated from positive samples of Campylobacter genus by MALDI-TOF MS. Our result showed that among the 150 positive strains isolated. 111 (74%) were specified as C. jejuni, 39 (26%) as C. coli and (0%) as C. lari.
 
Comparison of the results obtained by the molecular approach based on gyrB PCR-RFLP to those obtained by MALDI-TOF MS
 
Using RFLP for gyrB gene, five samples P13; P27; P48; P61; P65 were identified as being C. jejuni, and three (3) samples were identified as C. coli. Moreover, the gyrB RFLP-PCR failed to  identify  the  following  20  samples).  Sample P21 was identified as C. jejuni by MALDI-TOF MS method. The other 19 samples which were unidentified by the molecular approach, the MALDI-TOF-MS method identified  the P24; P47; P56; P64; P93; P102; P103; P104; P105; P106; P116; P120; P143; P162 samples as being C. jejuni while the samples P26; P33; P35; P51; P82 were identified as being C. coli. The results obtained from the different approaches has been summarised in Tables 2 and 3.
 


 DISCUSSION

Campylobacter was considered for a long time as part of the normal microbiota of birds and the infection of broiler chickens had not previously been considered to cause disease (Ishihara et al., 2016). Challenging this paradigm, a recent work proved that C. jejuni is not merely a commensal in commercial broiler chickens but have significant influence on animal health and welfare in intensive poultry production. The incidence of campylobacteriosis cases among humans has been shown to correlate with the prevalence of Campylobacter spp. among broiler chickens (Bahrndorff et al., 2013) humans with gastroenteritis (Masanta et al., 2016).
 
Efficient and consistent techniques for isolation and identification of Campylobacter species in poultry are crucial to enable epidemiological and clinical investigation. In this study, we have compared conventional and molecular methods to identify Campylobacter species in Moroccan poultry groups. Conventional methods based on morphological and biochemical proprieties are known to be relatively slow and laborious with a low discrimination power (Keramas et al., 2004). In our study also, 81% of the analysed samples (134/165 – Cf. Table 1), which were identified as Campylobacter genus, were unspecified because of their bacterial behaviour under conditions of antibiotics sensitivity reaction.
 
Biochemical tests carried out on strains whose morphological characteristics are suggestive of the genus Campylobacter make possible to identify the four major species of Campylobacter thermotolerants. Thus, the search for oxidase and catalase activity and the hippurate hydrolysis test are realized in association with the evaluation of sensitivity to two antibiotics, nalidixic acid and cephalotin. These tests are recommended by the ISO 10 272 standard (reference method for research thermotolerant Campylobacter in food). In practice, this biochemical identification of Campylobacter spp. presents difficulties of interpretation and some traps.
 
The hippurate hydrolysis test was widely used to distinguish thermotolerant Campylobacter species of C. jejuni (C. jejuni responds positively, C. coli and C. lari negatively), positive hippurate hydrolysis was pathognomonic to C. jejuni but the discovery of C. jejuni subsp jejuni strains negative to the hippurate test as C. lari strains (biotypes I and II) hippurate positive questions this method for the establishment of a formal distinction between   the    two  species  (Adzitey  and  Corry,
2011).
 
Also, C. jejuni strains resistant to nalidixic acid have appeared and pose problem for the use of antibiotic sensitivity as identification criteria (Wimalarathna et al., 2013).
 
As such, various methods based on nucleic acid amplification and sequencing has been proposed as an alternative to identify Campylobacter genus and the different species. These approaches determine specific thermophilic Campylobacter strains based on precise characterization of genomic DNA (Das et al., 2014) and included references.
 
 In the context of studying bacterial phylogeny and taxonomy, the use of 16S rRNA gene sequences has been by far the most common housekeeping genetic marker employed for several reasons.These reasons include its presence in almost all bacteria, often existing as a multigene family, or operons; the function of the 16S rRNA gene over time has not changed, suggesting that random sequence changes are a more precise measure of time (evolution); and the 16S rRNA gene (1500-bp) is large enough for bioinformatics analysis (Vergis et al., 2013). However, it was shown that 16S rRNA analysis enables specific identification of most Campylobacter species. The exception was a lack of discrimination among the taxa C. jejuni and C. coli and atypical C. lari strains, which shared identical or nearly identical 16S rRNA sequences (Loong et al., 2016). Indeed, the results of this study also confirmed the inability of the 16S rRNA sequencing procedure to differentiate these three species. In fact, the BLAST based comparisons to NCBI’s bacterial Sequence database provided a list of matching sequences that were a mixture of C. jejuni, C. coli and C. lari sequences, that possess identity scores of 100-98%. Knowing that suggested guidelines in literature describing the cut-off levels for sequence homology (percent identity) for proper identification of bacterial species ranging from ≥ 97% to ≥ 99% (Loong et al., 2016). Our results confirm the limitations of the 16S rRNA gene for resolving close relationship as previously demonstrated by Choi et al. (2016). The gyrB PCR-approach using DdeI as restriction enzyme provided a valuable tool for rapid and unambiguous identification of the majority of Campylobacter species. The analysis of our samples by gyrB PCR-RFLP showed a reliable discrimination between the three species in our collection. In fact, the 81% of our samples that failed to be characterized at species level with conventional methods have been well discriminated by gyrB PCR-RFLP. Nevertheless, the gyrB gene is not the only target gene with the power to identify pathogenic Campylobacter species. Other researches have shown that the lpxA and GTPase genes can also clearly identify and separate many of the Campylobacter species (Banowary et al., 2015; Klena et al., 2004).
 
With both methods, (gyrB RFLP-PCR and MALDI-TOF MS no strain was identified as C. lari. The misidentifications obtained with MALDI-TOF MS occurred only in samples where two strains coexisted. However, a discordance between gyrB RFLP-PCR and MALDI-TOF MS was found in 28 samples (Table 3). A total of 17 strains were identified as C. coli by gyrB RFLP-PCR and as C. jejuni by MALDI-TOF MS while 11 strains were specified as C. jejuni by gyrB RFLP-PCR and as C. lari by MALDI-TOF MS. For these strains, we have sequenced the gene encoding gyrB. All gyrB sequences of discordant strain when compared to the bacterial sequence database produced a collection of Campylobacter species sequences that were in perfect agreement with the results of the gyrB RFLP-PCR and that matched the query sequence with identity scores of 99-97%. However, the first match from the database that was in accordance with MALDI-TOF MS result had only a maximum identity of 90% (data not shown).


 CONCLUSION

Overall, this study demonstrates that Campylobacter spp. viscera colonization in the broiler chicken samples collected all over Morocco had a high prevalence (97%) of natural contamination with Campylobacter spp. throughout the 3-year period compared to the other results found in other developed and developing countries. Our study revealed that C. jejuni was a more frequent pathogen than C. coli and C. lari.
 
This study may contribute to establishing proper control, monitoring and management strategies from the farm through to the consumer in order to reduce the incidence of campylobacteriosis.


 CONFLICT OF INTERESTS

The authors have not declared any conflict of interests.



 REFERENCES

Adzitey F, Corry J (2011). A Comparison between Hippurate Hydrolysis and Multiplex PCR for Differentiating Campylobacter coli and Campylobacter jejuni. Trop. Life Sci. Res. 22:91-98.

 

Bahrndorff S, Rangstrup-Christensen L, Nordentoft S, Hald B (2013). Foodborne disease prevention and broiler chickens with reduced Campylobacter infection. Emerg. Infect. Dis. 19:425-430.
Crossref

 

Banowary B, Dang VT, Sarker S, Connolly JH, Chenu J, Groves P, Ayton M, Raidal S, Devi A, Vanniasinkam T, Ghorashi SA (2015). Differentiation of Campylobacter jejuni and Campylobacter coli using Multiplex-PCR and High Resolution Melt Curve Analysis. PLoS One, 10:e0138808.
Crossref

 

Barkok A (2008). Structure et importance des secteurs avicoles commercial et traditionnel au Maroc. Revue du secteur avicole. FAO/UNO. Translated into: Structure and importance of commercial and traditional poultry sectors in Morocco. Review of the poultry sector. FAO/UNO.

 

Begley M, Gahan CG, Hill C (2005). The interaction between bacteria and bile. FEMS Microbiol Rev. 29:625-651.
Crossref

 

Boelaert F, Amore G, Van der Stede Y, Hugas M (2016). EU-wide monitoring of biological hazards along the food chain: achievements, challenges and EFSA vision for the future. Recent Curr. Opin. Food Sci. 12:52-62.
Crossref

 

Branda JA, Rychert J, Burnham CA, Bythrow M, Garner OB, Ginocchio CC, Jennemann R, Lewinski MA, Manji R, Mochon AB (2014). Multicenter validation of the VITEK MS v2.0 MALDI-TOF mass spectrometry system for the identification of fastidious gram-negative bacteria. Diagn. Microbiol. Infect. Dis. 78:129-131.
Crossref

 

Cean A, Stef L, Simiz E, Julean C, Dumitrescu G, Vasile A, Pet E, Drinceanu D, Corcionivoschi N. (2015). Effect of human isolated probiotic bacteria on preventing Campylobacter jejuni colonization of poultry. Foodborne Pathog Dis, 12:122-130.
Crossref

 

Choi HS, Shin SU, Bae EH, Ma SK, Kim SW (2016). Infectious Spondylitis in a Patient with Chronic Kidney Disease: Identification of Campylobacter fetus subsp. testudinum by 16S Ribosomal RNA Sequencing. Jpn. J. Infect. Dis. 69:517-519.
Crossref

 

Das S, Dash HR, Mangwani N, Chakraborty J, Kumari S (2014). Understanding molecular identification and polyphasic taxonomic approaches for genetic relatedness and phylogenetic relationships of microorganisms. J. Microbiol. Methods, 103:80-100.
Crossref

 

De Carolis E, Vella A, Vaccaro L, Torelli R, Spanu T, Fiori B, Posteraro B, Sanguinetti M (2014). Application of MALDI-TOF mass spectrometry in clinical diagnostic microbiology. J. Infect. Dev. Ctries. 8:1081-1088.
Crossref

 

Di Giannatale E, Garofolo G, Alessiani A, Di Donato G, Candeloro L, Vencia W, Decastelli L, Marotta F (2016). Tracing Back Clinical Campylobacter jejuni in the Northwest of Italy and Assessing Their Potential Source. Front Microbiol. 7:887.
Crossref

 

EFSA (2011). Scientific Opinion on Campylobacter in broiler meat production: control options and performance objectives and/or targets at different stages of the food chain. EFSA J. 9(4):2105 [141 pp].
Crossref

 

Gonsalves CC, Borsoi A, Perdoncini G, Rodrigues LB, do Nascimento VP (2016). Campylobacter in broiler slaughter samples assessed by direct count on mCCDA and Campy-Cefex agar. Braz. J. Microbiol. 47:764-769.
Crossref

 

Humphries RM, Schuetz AN (2015). Antimicrobial susceptibility testing of bacteria that cause gastroenteritis. Clin. Lab. Med. 35:313-331.
Crossref

 

Ishihara K, Chuma T, Andoh M, Yamashita M, Asakura H, Yamamoto S (2016). Effect of climatic elements on Campylobacter colonization in broiler flocks reared in southern Japan from 2008 to 2012. Poult. Sci. pew354.
Crossref

 

Kamei K, Asakura M, Somroop S, Hatanaka N, Hinenoya A, Nagita A, Misawa N, Matsuda M, Nakagawa S, Yamasaki S (2014). A PCR-RFLP assay for the detection and differentiation of Campylobacter jejuni, C. coli, C. fetus, C. hyointestinalis, C. lari, C. helveticus and C. upsaliensis. J. Med. Microbiol, 63:659-666.
Crossref

 

Kawasaki S, Fratamico PM, Wesley IV, Kawamoto S (2008). Species-specific identification of Campylobacters by PCR-restriction fragment length polymorphism and PCR targeting of the gyrase B gene. Appl. Environ. Microbiol. 74:2529-2533.
Crossref

 

Keramas G, Bang DD, Lund M, Madsen M, Bunkenborg H, Telleman P, Christensen CB (2004). Use of culture, PCR analysis, and DNA microarrays for detection of Campylobacter jejuni and Campylobacter coli from chicken feces. J. Clin. Microbiol. 42:3985-3991.
Crossref

 

Kittitrakul C, Lawpoolsri S, Kusolsuk T, Olanwijitwong J, Tangkanakul W, Piyaphanee W. (2015). Traveler's Diarrhea in Foreign Travelers in Southeast Asia: A Cross-Sectional Survey Study in Bangkok, Thailand. Am. J. Trop. Med. Hyg. 93:485-490.
Crossref

 

Klena JD, Parker CT, Knibb K, Ibbitt JC, Devane PM, Horn ST, Miller WG, Konkel ME (2004). Differentiation of Campylobacter coli, Campylobacter jejuni, Campylobacter lari, and Campylobacter upsaliensis by a multiplex PCR developed from the nucleotide sequence of the lipid A gene lpxA. J. Clin. Microbiol. 42:5549-5557.
Crossref

 

Loong SK, Khor CS, Jafar FL, AbuBakar S (2016). Utility of 16S rDNA Sequencing for Identification of Rare Pathogenic Bacteria. J. Clin. Lab. Anal. 30:1056-1060.
Crossref

 

MacDonald E, White R, Mexia R, Bruun T, Kapperud G, Lange H, Nygard K, Vold L (2015). Risk Factors for Sporadic Domestically Acquired Campylobacter Infections in Norway 2010-2011: A National Prospective Case-Control Study. PLoS One, 10:e0139636.
Crossref

 

Masanta WO, Lugert R, Gross U, Linsel G, Heutelbeck A, Zautner AE. (2016). Seroprevalence of Campylobacter-Specific Antibodies in two German Duck Farms - A Prospective Follow-Up Study. Eur. J. Microbiol. Immunol. 6:118-123.
Crossref

 

Merritt ME, Donaldson JR. (2009). Effect of bile salts on the DNA and membrane integrity of enteric bacteria. J. Med. Microbiol. 58:1533-1541.
Crossref

 

Rahi P, Prakash O, Shouche YS (2016). Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass-Spectrometry (MALDI-TOF MS) Based Microbial Identifications: Challenges and Scopes for Microbial Ecologists. Front. Microbiol. 7:1359.
Crossref

 

Rodgers JD, Simpkin E, Lee R, Clifton-Hadley FA, Vidal AB. (2016). Sensitivity of Direct Culture, Enrichment and PCR for Detection of Campylobacter jejuni and C. coli in Broiler Flocks at Slaughter. Zoonoses Public Health.

 

Samosornsuk W, Asakura M, Yoshida E, Taguchi T, Eampokalap B, Chaicumpa W, Yamasaki S (2015). Isolation and Characterization of Campylobacter Strains from Diarrheal Patients in Central and Suburban Bangkok, Thailand. Jpn. J. Infect. Dis. 68:209-215.
Crossref

 

Umaraw P, Prajapati A, Verma AK, Pathak V, Singh VP (2017). Control of Campylobacter in poultry industry from farm to poultry processing unit: A review. Crit. Rev. Food. Sci. Nutr. 57:659-665.
Crossref

 

Vergis J, Negi M, Poharkar K, Das DP, Malik SV, Kumar A, Doijad SP, Barbuddhe SB, Rawool DB. (2013). 16S rRNA PCR followed by restriction endonuclease digestion: a rapid approach for genus level identification of important enteric bacterial pathogens. J. Microbiol. Methods 95:353-356.
Crossref

 

Vondrakova L, Pazlarova J, Demnerova K (2014). Detection, identification and quantification of Campylobacter jejuni, coli and lari in food matrices all at once using multiplex qPCR. Gut Pathog. 6:12.
Crossref

 

Weisburg WG, Barns SM, Pelletier DA, Lane DJ. (1991). 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173:697-703.
Crossref

 

Wimalarathna HM, Richardson JF, Lawson AJ, Elson R, Meldrum R, Little CL, Maiden MC, McCarthy ND, Sheppard SK (2013). Widespread acquisition of antimicrobial resistance among Campylobacter isolates from UK retail poultry and evidence for clonal expansion of resistant lineages. BMC Microbiol, 13:160.
Crossref

 

WHO - World Health Organization - Fact sheet : Campylobacter (December 2016). 

View

 




          */?>