Biotyping of Multidrug Resistant Campylobacter Jejuni from Poultry and Humans in Northern Region of Ghana

Campylobacters are zoonotic bacteria which are universally famous for causing enteritis in humans. They are normally found as commensals in the digestive tract of food animals with poultry being a major reservoir of the pathogenic species. This study evaluated the presence of Campylobacter in poultry (commercial and domestic) and humans (patients and asymptomatic individuals) and characterized strains by biotyping and susceptibility test in the Northern region of Ghana.


Abstract
Background Thermophilic Campylobacters are zoonotic bacteria which are universally famous for causing enteritis in humans. They are normally found as commensals in the digestive tract of food animals with poultry being a major reservoir of the pathogenic species. This study evaluated the presence of Campylobacter in poultry (commercial and domestic) and humans (patients and asymptomatic individuals) and characterized strains by biotyping and susceptibility test in the Northern region of Ghana.

Results
Of the 346 poultry and 741 human samples analyzed, 43.1% and 12.9% Campylobacter species were recovered. Isolation frequency from commercial birds was signi cantly more (50.5%) than domestic birds (33.8%), as incidence in patients and asymptomatic individuals was 15.4% and 9% with a p=0.001. Campylobacter jejuni were recovered from more than eighty percent (84%) of commercial birds and 64% of domestic birds and in humans signi cantly less strains were observed in patients than asymptomatic individuals (p <0.05). Distribution of biotypes in domestic birds, patients and asymptomatic individuals were comparable with C. jejuni biotype I prevalence and Bioytype II largely in commercial poultry. All strains were resistant to tetracycline; and against erythromycin, 69.3% of jejuni strains were resistant but no resistance was recorded among the non-jejuni strains. Most resistance was noted among patient strains. Imipenem and the aminoglycosides were relatively effective as resistance of 10% and below 20% were obtained. Multidrug resistance was between 94-100% with 60% of strains being resistant to 4 or 5 classes of antibiotics.

Conclusion
This study found phenotypic correlation between poultry and human strains coupled with copious isolation of C. jejuni biotypes I and II from commercial and domestic poultry which may well be possible source of threat to public health. None of the endorsed treatment drugs (erythromycin, cipro oxacin, and tetracycline) can be admitted in this region due to common resistance found among strains against these agents but imipenem and aminoglycosides proved to be reliable as strains showed ample susceptibility.
The multiple drug resistance (94-100%) observed in our study and other related investigations in the country call for rapid implementation of the policy on antimicrobial use and resistance which has been launched in Ghana.

Background
Campylobacter are Gram-negative zoonotic pathogens of global signi cance, especially the thermophilic species. Of the three species (jejuni, coli, lari), Campylobacter jejuni is crucial and liable for colonic colonization in poultry and food-borne enteritis in humans. Generally, C. jejuni is non-pathogenic in poultry though freshly hatched chickens are susceptible to ephemeral diarrhea following infection.
Consumers of undercooked poultry meat, beef, pork and poultry products may be at risk of campylobacteriosis which is a major cause of human enteritis [1].
The importance of campylobacteriosis as a foodborne pathogen is established by surveillance studies reporting 15918 hospitalizations in the UK [2], 2, 00,507 cases in the European Union [3] and an incidence of 12.71 cases per 100.000 inhabitants in USA [4]. Inadequate systematic investigations to detect outbreaks whose strains could serve as basis for source attribution and risk valuation make it hard to establish factual incidence of the infection in developing countries [5]. But campylobacteriosis is viewed as hyper-endemic in many developing regions because of numerous factors including poor environmental sanitation, proximity with animals at native settings in rural, peri urban and farming communities [6]. Marquis and colleagues recount live, unrestricted birds kept in close association with humans as key potential source of C. jejuni infection [7]. Likewise in the UK, 97% of sporadic infections have been linked to farmed animals [8]. The degree to which poultry intake is responsible for human infections is however not precisely known [9].
Resistance trends and biotyping have been used in epidemiological investigations to track potential sources or supposed routes of campylobacteriosis outbreaks. Such studies have shown prevalence of different biotypes of species oscillating between 40-100% in birds including chicken [10,11]. Semblance of Campylobacter species recovered from human patients and poultry proposes commonality of infection according to Bruce et al. [12]. This has been validated by some studies which have shown related types of Campylobacter from poultry and humans [13,14].
Antimicrobial therapy in campylobacteriosis is generally recommended in severe cases and immunesuppressed individuals in which case macrolides ( rst choice) and uoroquinolones (second choice) are considered [15]. These drugs were previously reported to be effective but misuse of antibiotics in both veterinary and human medicine have contributed to escalated incidence of resistant strains documented worldwide [16].
In evaluating the threat associated with the presence of Campylobacter in poultry, it is an essential step to identify phenotypic characters that provide epidemiological markers in both animal and human population. The Northern region of Ghana is an agricultural region with majority of the people engaged in animal husbandry but research on zoonotic infections including campylobacteriosis is dolefully inadequate. To provide a baseline information on this important pathogen, the study determined the presence of Campylobacter in the cloaca of poultry and the stools of symptomatic and asymptomatic individuals, assessed the resistance patterns of species and compared the biotypes of C. jejuni in humans and poultry.

Resistance of Campylobacter jejuni and non-jejuni species
All (100%) the non-jejuni strains were susceptible to erythromycin while 69.4% of C. jejuni were resistant but against trimethoprim sulphamethoxazole (SXT), nalidixic acid and chloramphenicol, non-jejuni strains showed more resistance which was signi cant, P = 0.001. Drugs which recorded more C. jejuni resistance included cipro oxacin, nor oxacin, amikacin and imipenem, Fig. 3.

Resistance pattern of Campylobacter jejuni biotypes in poultry and humans
Most resistance to erythromycin (82.8%) was found in Biotype I. Biotype II strains generally showed lower resistance, with exception to cipro oxacin and nor oxacin where their resistance were more. Resistance of Biotype II to chloramphenicol was below 10% but up to 52% rate was recorded among Biotypes I and III strains. Highest resistance to trimethoprim sulphamethoxazole was observed among Biotype III strains, Fig. 4. The only two isolated strains of Biotype IV were resistant to ceftriaxone but susceptible to erythromycin, nalidixic, nor oxacin, imipenem and the aminoglycosides. With the exception of chloramphenicol, resistance difference among biotypes was not statistically signi cant. Multidrug resistance was 94.7% in poultry and 100% among human strains. Sixty percent (60%) of poultry and human strains were resistant to 4 or 5 classes and up to about 37% to 6 or 7 classes of antibiotics, Table 3.

Discussion
Considering potential risks associated with Campylobacter in poultry, it is critical to identify these strains in the human population. This study isolated and characterized Campylobacter from poultry and humans and respectively found prevalence of 43.1% and 12.9% which is higher in the case of poultry but slightly lower in reference to human, than reported rates in the southern region of the country [17,18]. Elsewhere in Canada, comparable rate of 40% was reported in poultry while divergent results of 62%, 87.2% and 90% were recorded in Morocco, Poland and Cameroon [14,[19][20][21]. A review of most studies across Africa reporting on Campylobacter isolation from stools generally documented low rates [22], which could be attributed to the isolation method that is typically culture rather than PCR which is superior and more sensitive in detection. Recovery of Campylobacter from patients with enteritis is usually greater than asymptomatic individuals as shown in our study where 15.4% and 9% were respectively recorded with supporting reports of 55% rate among patients in Nigeria, 16.6% from individuals in contact with foodproducing animals in central Egypt and 11.4% from individuals in eastern Tanzania [23][24][25].
Campylobacter jejuni dominance (80%) in poultry was normal, conforming to literature reports as the most common species found in poultry and its products. In humans however, more non-jejuni species were recovered and the explanation could be the possible acquisition from different sources such as other animals (livestock) and the environment where these non-jejuni strains are rifer. It was intriguing to nd more non-jejuni strains in the domestic birds than the commercial birds which could be credited to the free access of domestic birds to the environment and other animals where they could have picked the strains.
Biotyping of C. jejuni was explored to reveal the relatedness of poultry and human strains and to provide epidemiological indicators in the study of Campylobacter infections in this region. The distribution of biotypes in patients, asymptomatic individuals and domestic birds were similar with Biotype I incidence while analogous ndings have been described in Australia, France, Iran and Nigeria [13,[26][27][28]. A study by Nadeau and colleagues discovered almost all invasive strains of C. jejuni belonged to biotypes I and II as cell-cytotoxic isolates were associated with III and IV and further suggested that clonally related isolates have common in vitro virulence characteristics [29]. In this study, the phenotypic relationship between poultry and human strains as well as the high proportion of C. jejuni biotypes I and II in both commercial and domestic poultry is a potential source of threat for humans as infections from these biotypes could be invasive and consequently dire. But, further studies on virulence factors and an assessment of the outcomes of infections caused by these biotypes would give a better understanding of this risk, since invasive properties of C. jejuni strains can in uence the austerity of clinical changes [1].
Routine treatment for Campylobacter infection is erythromycin but uoroquinolones (speci cally cipro oxacin) and tetracycline are alternatively prescribed. Susceptibility to tetracycline was 0% in both poultry and human strains. This results was not startling because tetracycline which is an over the counter drug is readily available and among the popular drugs consumed in both veterinary and clinical practice in Ghana. It is frequently used by commercial poultry farmers and well identi ed by rural domestic breeders as prophylaxis and treatment drug for poultry infections. It is also habitually prescribed in hospitals to manage enteritis and other conditions (Personal communication).
Resistance to erythromycin was only found in C. jejuni as 100% susceptibility was detected among nonjejuni strains. Although some countries have recorded low and stable rates of resistance to erythromycin [30,31] studies in Ghana and some other African countries have observed contradictory results [32,33,17,18]. Erythromycin as a drug of choice has performed poorly against human and animal Campylobacters in Ghana as evidenced in previous studies in a different region of the country [17,18]. Some authors have predicted increased risk of adverse events including Guillain-Barré syndrome and severe reactive illnesses with macrolide-resistant Campylobacters [34]. Other investigations also predict an association of macrolide resistance in clinical C. jejuni strains with some virulence markers and so the use of it in any ecology may select for such strains [34]. Thus, occurrence of macrolide resistant Campylobacter could be of public health signi cance even in the absence of proof of treatment failure [35]. The carbapenems (meropenem, imipenem) are among the last line drugs often used in the management of infections caused by multidrug resistant pathogens in Ghanaian hospitals (Personal communication). High susceptibility of up to 100% were previously reported [17,18] but recent studies indicate increasing resistance though not at alarming levels (10%).
Multidrug resistance was 94.7% in poultry and 100% in humans with 60% of isolates showing resistance to four or ve drugs. Multiple drug resistance invariably leads to expensive health care cost with attendant nancial drain particularly on the poor and average patient in a Low Middle Income Country. The fallout of antimicrobial resistance in animals and humans is an emphasis of commonly used and abused drugs. This became evident in our study where resistance of 10% and below 20% were observed against imipenem and the aminoglycosides. Because these drugs are used to manage severe and complicated infections they are infrequently prescribed and also not readily available for potential abuse.
In view of these, antibiotic resistance surveillance studies in animal, human and environment have become obligatory especially in under resourced regions of the country as most studies are focused in the well-resourced regions. Data from such investigations will inform stakeholders and buttress the need for the implementation of the national policy on antimicrobial use and resistance.

Conclusion
The study found phenotypic linkages between poultry and human strains with C. jejuni Biotype I and II dominance, that might pose some threat to human through consumption of contaminated poultry to which further studies are recommended to establish the extent of risk. All isolated Campylobacter species were resistant to tetracycline as patient strains were commonly resistant. None of the endorsed treatment drugs (Erythromycin, tetracycline and cipro oxacin) for Campylobacter infections may be empirically safe in this region without susceptibility con rmation from the laboratory. The carbapenems (Imipenem) and the aminoglycosides (amikacin, gentamicin) on the other hand appeared effective in-vitro. The recorded MDR of 95-100% re-echo the urgency to implement the national policy on antimicrobial use and resistance before the situation becomes unmanageable.

Study Area
This research was conducted in the Tamale metropolis and Tolon district of the Northern Region. Tamale, the capital of the Region is the third largest city in Ghana and located 600 km north of Accra, the capital of the country. Sampling sites included the Tamale Teaching Hospital (TTH), Tamale Central Hospital (TCH), six (6)  Availability of data and materials We consider our data private but the corresponding author will make it available upon reasonable request.

Competing interests
The authors declare that they have no competing interests Funding Financial support was received from Faculty of Science, University of Helsinki for sample collection.  Biotype I n=29 Biotype II n=31 Biotype III n=10