Antimicrobial Susceptibility and Serovars of Salmonella from Chickens and Humans in Ibadan, Nigeria

Background: This study determines the prevalence and antibiotic resistance of Salmonella serovars from humans and chickens in Ibadan, Nigeria, in 2004-2007. Methodology: A total of 991 blood samples were collected from patients in 2004 to 2005 and 641 fecal samples were collected from poultry farms in 2007. All Salmonella isolates were serotyped and tested for antimicrobial susceptibility. Results: Thirty-nine (4%) Salmonella isolates were obtained from human blood and 70 (11%) from chicken fecal samples. The human isolates revealed nine different serovars; 82% were non-typhoidal Salmonella and 18% were (S. Typhi). The majority of serovars from humans were S. trimethoprim, and ampicillin ranged from 36% to 59% for the human isolates. Eight different serovars were obtained from chickens; S. Virchow (71%) predominated. A high frequency (87%) of reduced susceptibility to ciprofloxacin was observed among the chicken isolates. A high frequency of resistance to tetracycline (93%), nalidixic acid (81%), and sulfamethoxazole (87%) was observed. Rare serovars such as S. Apapa, S. Mouschaui, S. Jukestown, S. Oritamerin, and S. Onireke were isolated from both humans and chickens. Identical serovars were not found among human and chicken isolates. Conclusions: This study indicates that chickens are not a reservoir of Salmonella causing bacteraemia among humans in Ibadan, Nigeria. Studies locating the reservoirs responsible for invasive salmonellosis in humans are needed. Controls and targeted interventions against S. Virchow and the frequent occurrence of antimicrobial resistance in chickens should be initiated to prevent the spread of this serovar.


Introduction
Salmonella enterica is an important food-borne pathogen that is currently divided into 2,587 serotypes [1].The typhiodal Salmonella serovars are host-restricted human pathogens and include Salmonella enterica serotype Typhi and S. Paratyphi [2].Non-typhoidal Salmonella (NTS) are zoonotic agents and a wide variety of animals have been identified as reservoirs [3][4][5].Poultry are considered as one of the most common vehicles of human salmonellosis [6].
Salmonellosis is often associated with gastroenteritis, which is usually self-limiting.In some cases, particularly in children, the elderly, and immunocompromised patients, Salmonella infection can lead to invasive and focal infections that can be severe [7].The ability of Salmonella to cause invasive infection varies with the serovar, the age of patient, and region.S. Typhi and S. Paratyphi A are highly invasive for both children and adults.Certain serovars of NTS, such as S. Choleraesuis, S. Dublin, and S. Virchow, have higher abilities than other serovars to cause invasive salmonellosis [8][9][10].In sub-Saharan Africa, NTS invasive infections are a public health concern for infants, young children, and young adults suffering from malnutrition, malaria, and HIV infection [11][12][13].Most human infections are caused by a limited number of serovars, which may vary from country to country and over time.The predominant serovars in some African countries include S. Enteritidis, S. Typhimurium, S. Concord, and S. Isangi [10,[12][13][14][15][16].
In Nigeria, there is limited data of the prevalence in Salmonella serovars causing human salmonellosis and the importance of transmitting food-borne pathogens from the food chain to humans is not well understood.Decades ago, a study reported the important role of S. Typhi and NTS in septicemia in humans in Ibadan, Nigeria; however, the specific NTS serotypes [17] were not revealed, probably due to the lack of resources and high-quality antisera that are often lacking from many African countries [18].Chicken is an important food source to man; chicken meat dishes are special delicacies, particularly during festive periods in Nigeria, but the poultry industry is largely unregulated.The link between human salmonellosis and consumption of chicken is not known nor is the prevalence of the serovars in Nigeria.A previous study found the prevalence of S. Enteritidis in chicken meat in Maiduguri, Nigeria, to be 27% [19].A recent study from Maiduguri, in the northern part of Nigeria, reported S. Hiduddify to be the predominant serovar in free-range chickens and poultry meat [20].
Treatment with antimicrobials is crucial for the proper management of severe or invasive human salmonellosis.Fluoroquinolones and third-generation cephalosporins are now commonly used in adults for treatment due to widespread resistance to chloramphenicol, ampicillin, and cotrimoxazole [21].Fluoroquinolones are often the last resort for treatment of children and are listed by the World Health Organization as critically important antimicrobials for human health [7,22].Multi-drug resistance and reduced susceptibility to ciprofloxacin have been reported from some African countries.Increasing multi-drug resistance associated with NTS in humans has been reported from Malawi and Ethiopia [15,23]; multi-drug resistance rates of 10% or less with decreased susceptibility to ciprofloxacin have also been reported from Senegal [14].A recent study reported high rates of resistance to ciprofloxacin in S. Hiduddify in chickens in Maiduguri, Nigeria [20]; otherwise, there are limited data on antimicrobial resistance on Salmonella in both humans and food animals in Nigeria.
This study was conducted to determine the prevalence of Salmonella serovars in chickens and from invasive extraintestinal salmonellosis in humans from the city of Ibadan, Nigeria, in various periods between 2004 and 2007.The susceptibility to antimicrobial resistance in the Salmonella isolates was also investigated and the presence of qnr genes was investigated in the isolates with a resistance phenotype indicating the presence of qnr genes.The serovars and susceptibility patterns of the isolates from the different reservoirs were compared.

Isolation of bacteria from humans
During 2004 and 2005, blood samples were collected from 991 febrile patients attending seven major hospitals and a private diagnostic laboratory in Ibadan province.Blood samples of 10 to 15 mL and 2 to 5 mL of were collected from adults and children, respectively.Bacteria were isolated from each blood sample following standard procedure by using Brain-Heart infusion broth (Lab M, Lancashire, UK) followed by plating on Brain-Heart Infusion agar supplemented with 5% sheep blood, Salmonella-Shigella agar (Lab M, Lancashire, UK), and McConkey agar (Lab M, Lancashire, UK).The isolates were incubated aerobically at 37 °C for 18 to 24 hours [24].Presumptive Salmonella colonies were identified by standard methods [25,26].

Isolation of bacteria from chicken
In 2007, a total of 641 fecal samples were collected at 12 intensive poultry farms.Five grams of each chicken fecal sample were enriched in 25 mL of selenite-F broth (Lab M, Lancashire, UK) and incubated aerobically at 37 °C for 18 to 24 hours followed by plating on Salmonella-Shigella agar (Lab M, Lancashire, UK) and incubated aerobically at 37 °C for 18 to 24 hours.Presumptive Salmonella colonies were identified following standard methods [25,26].

Statistical analysis
SAS version 9.1.3(SAS Institute Inc.) was used to assess the association of the prevalence in males compared to the prevalence in females using a Fisher's Exact test.The criteria for evaluating the significance level in the model was a P-value of < 0.05.

Serotyping
Serotyping of all the presumptive biochemical positive Salmonella isolates was performed at the National Salmonella and Shigella Centre, Bangkok, Thailand, according to the previously described method [27].

Antimicrobial susceptibility testing
Susceptibility to antimicrobial agents was performed at the National Food Institute, Copenhagen, Denmark (DTU-FOOD) on all Salmonella isolates as minimum inhibitory concentration (MIC) determinations according to the previously described method [27].In addition to the previously described method, the following antimicrobial and resistance epidemiological cut-off values were used in the study for cefotaxime; (R > 2 mg/mL) (www.eucast.org).

Detection of plasmid mediated quinolone resistance genes
Strains showing reduced susceptibility to ciprofloxacin and susceptible to nalidixic acid were further characterized through the use of a PCR assay with primers specific for qnrA, qnrB, qnrC, qnrD, qnrS, and aac(6')-lb [23,28,29].PCR products were purified using the GFX™ PCR DNA kit (GE Healthcare, Chalfont St. Giles, UK) and submitted to Macrogen Inc. for sequencing.Sequence analysis and alignment was performed using the Vecton NTI suite 9 (InforMax Inc., Bethesda, Maryland, US) and the resulting nucleotide sequences were compared to sequences obtained from GenBank (http://www.lahey.org/studies/webt.html).

Bacterial isolates from humans
Of the 991 blood samples collected, 519 samples originated from males.Thirty-nine samples yielded Salmonella, which gave an overall prevalence among humans submitting a blood sample at 4%.The prevalence ranged substantially between the different sample sites from 1% (diagnostic laboratory) to 10% (hospital B) (Table 1).The prevalence between males and females differed with 5% among males in contrast to 3% among females (not significant p = 0.07).The overall age of patients from whom the positive blood samples were collected ranged from seven days to 28 years; however, large differences were observed between the different hospitals: 7 days to 4 years in hospital A; 3 months to 2 years and 6 months in hospital B; 18 days to 2 years in hospital C; 2 years and 5 months to 7 years in hospital D; 1 year and 7 months to 10 years and 7 months in hospital E; 1 year and 3 months to 16 years in hospital F; 11 years to 28 years in hospital G; and 14 years at the diagnostic laboratory.The median age of the febrile patients that yielded Salmonella was 1 year and 7 months.

Bacterial isolates from chickens
Out of the 641 chicken fecal samples collected, 70 were positive for Salmonella, giving an overall prevalence of 11%.The number of samples investigated and the number of samples positive for Salmonella fluctuated among the different farms, resulting in a prevalence ranging from 2% (farm C and G) to 26% (farm J) (Table 2).
Hospitals A and B accounted for 21 of the isolates.These two hospitals also accounted for four out of seven (57%) S. Dublin, 10 out of 13 (77%) S. Enteritidis, and six out of seven (86%) S. Typhimurium (Table 3).None of the serovars found in humans were observed among the isolates from chicken (Tables 3 and 4).

Serotyping of chicken isolates
The 70 Salmonella isolates revealed eight different serovars with S. Virchow being the most frequent, accounting for 71% (n = 50) of all serovars isolated from chickens.S. Virchow was found in chickens from 11 of the 12 farms, accounting for 92% (n = 22) and 83% (n = 10) of all serovars found on farms J and K. Additionally, it was the only serovar originating from chickens in farms B, C, and D (Table 2).Two uncommon serovars from chickens were isolated: S. Haifa (n = 2; 3%) and S. Onireke (n = 1; 1%) (Table 2).

Antimicrobial susceptibility testing of human isolates
The Salmonella isolates originating from humans were all susceptible to apramycin, ceftiofur, colistin, florfenicol, cefotaxime, gentamicin, and neomycin.Low frequency of resistance was observed to nalidixic acid (3%) and ciprofloxacin (3%).
Antimicrobial resistance to spectinomycin, chloramphenicol, tetracycline, and streptomycin ranged from 18% to 44%.A higher frequency of antimicrobial resistance was observed to sulfamethoxazole (54%), trimethoprim (54%), amoxicillin+clavulanic (54%), and ampicillin (59%) (Table 4).Three of the seven S. Dublin were resistant to ampicillin, streptomycin, sulfamethoxazole, tetracycline, and trimethoprim; the remaining four isolates were pansusceptible.Antimicrobial resistance was observed in eleven of the 13 S. Enteritidis isolates.Four S. Enteritidis isolates conferred resistance to ampicillin, chloramphenicol, sulfamethoxazole, and trimethoprim.Two additional isolates also conferred resistance to streptomycin.One of the remaining five S. Enteritidis isolates was resistant to ampicillin, trimethoprim, streptomycin, amoxicillin+clavulanic acid, nalidixic acid, and tetracycline.None of the 13 S. Enteritidis isolates were pansusceptible (data not shown).Six of seven S. Typhi isolates were pansusceptible while the remaining one isolate was resistant only to streptomycin.

Detection of qnr genes
Four S. Derby isolates from chickens showed reduced susceptibility to ciprofloxacin and were susceptible to nalidixic acid.All four harbored the qnrS1 gene.The qnrS1 positive S. Derby were from farm K (n = 1), farm I (n = 2), and farm F (n = 1).

Discussion
In this study, the overall positive isolation rate of Salmonella species from human blood samples is comparable with that from Ethiopia (4%) [30] but higher than those from Kenya (1%) [31] and Mozambique (2%) [32].On the other hand, higher rates have been reported from Congo (7%) [33] and Malawi (6% and 8%) [15,34].The skewed distribution of 1:0.5 between males and females is difficult to explain, but may be caused by the fact that more males were examined or that the male population has a higher susceptibility to infections.
Our data reveals the relative importance of Salmonella associated with human bacteraemia predominated by S. Enteritidis and S. Typhimurium in Ibadan, Nigeria.Similar trends have been observed in African countries where S. Enteritidis and S. Typhimurium are the most common serovars causing human salmonellosis [9,11,13,15,[35][36][37][38][39][40].It is interesting to note that the majority of the S. Enteritidis and S. Dublin were isolated from hospitals A, B, and C, whereas the remaining hospitals found very few.S. Typhimurium was mostly isolated from hospital B. Kariuki     compared to other non-typhoidal Salmonella [38].Similar trends have been observed elsewhere [7,10].
Epidemic increase in prevalence of S. Typhimurium has recently been linked to circulation of a particular MLST clone, ST313, in sub-Saharan African countries [13]; however, it has not been determined if the S. Typhimurium ST313 clone has spread to Ibadan, Nigeria.A future study will reveal if this is the case or if the isolates belong to the previously described clone of phage type U282 [39].Similarly, the clonality of invasive S. Enteritidis also needs to be investigated to determine whether certain clones are responsible for causing bacteraemia compared to those causing mild gastroenteritis.It is also noteworthy that neither S. Enteritidis nor S. Typhimurium was isolated from chickens included in this study, indicating that intensively raised chickens are not reservoirs for invasive serovars in humans.It should be noted, however, that the samples obtained from chickens were collected in 2007, compared to the samples from humans, which were collected from 2004 to 2005.In 2004, S. Enteritidis and S. Typhimurium were isolated by Orji et al. from poultry droppings and beef [41].
S. Dublin was not reported as an important cause of invasive salmonellosis in some sub-Saharan African countries [9,11,13,15,[35][36][37][38], but this serovar constitutes an important human pathogen in Upper Volta [42].S. Dublin is host-adapted to cattle and is highly invasive; it is transmitted to humans through beef, cheese, and raw milk [43][44][45].In many European countries, surveillance shows that S. Dublin is the most commonly isolated serovar from bovine meat, exceeding the level of S. Typhimurium [46].Only a few publications have described S. Dublin in animals from Nigeria and all of them also associate the serovar to cattle [47,48].Cattle farming is a thriving business in Nigeria because beef meat is highly consumed, either in soup dishes or roasted (suya); other products such as raw milk and cheese are heavily consumed in Nigeria without adequate control measures to prevent spread of zoonotic agents to human.It would be interesting to determine the occurrence of S. Dublin among cattle in Nigeria.
The study further reveals that S. Typhi still constitutes a significant public health importance in Ibadan, Nigeria; therefore, appropriate control measures, such as vaccination and food safety measurements, need to be put in place.Overall, the study supports the conclusions of other publications which report that invasive salmonellosis caused by NTS is of greater public health importance than the S. Typhi [9].
Rarely isolated Salmonella serovars such as S. Apapa, S. Mouschaui, S. Jukestown, and S. Oritamerin were observed causing invasive salmonellosis in humans.Severe human salmonellosis caused by those serovars has been previously linked to exposure to reptiles and amphibians [49][50][51][52][53][54].Several studies have shown that reptiles are reservoirs for Salmonella that are able to affect humans and recently constitute a significant public health problem in many countries due to captive pet reptiles [55][56][57]; however, the majority of infections in Nigeria are not caused by captive pet reptiles but more likely by household wall lizards.Lizards are found around houses in Nigeria, most commonly in the rural areas and sometimes inside houses, thus constituting a potential health risk.Additionally, snake meat is a special delicacy in some parts of Nigeria, while snakes are revered in other places.It is interesting to note that all the rarely isolated serovars obtained, with the exception of S. Apapa, were isolated from samples at more rural sampling sites, thus suggesting that the sources of infections were associated with reptiles or the environment.Wall geckos have been previously described as a reservoir of Salmonella in Nigeria, constituting a public health problem [58,59] that is difficult to control due to the nature of wild animals.Consequently, the potential risk of invasive salmonellosis due to the exposure to reptiles and amphibians should be disseminated to the people at risk in rural areas.
The majority of the human isolates were resistant to ampicillin, sulfamethoxazole, and trimethoprim; high resistance was also observed to chloramphenicol.Multidrug resistance to these old and commonly used antimicrobials is a public health problem [60] and could facilitate usage of fluoroquinolones and third-generation cephalosporins for empiric treatment.S. Typhimurium was the most resistant of the serovars obtained from human blood samples and exhibited resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, tetracycline, and trimethoprim.In many ways, the S. Typhimurium isolates in this study match the resistance profile of the multidrug-resistant invasive S. Typhimurium ST313 clone found in sub-Saharan Africa [13].This hypothesis needs to be further investigated by MLST typing before it is concluded.
It is interesting that all the isolates of S. Typhi were susceptible to all the tested antibiotics, suggesting the efficacy of old antimicrobials for treatment of typhoid fever.
The overall prevalence rate (11%) of Salmonella spp.from chicken fecal samples is high and with prevalence rates of 26% and 15% in farms J and K, respectively.Similarly, high prevalence rates have also been reported from Maiduguri, northern Nigeria [20], and a high rate (37%) of Salmonella contamination of broiler farms has been reported from Algeria [61].Chicken is also a predominant source of Salmonella in Europe and has warranted initiation of targeted control measures [46,62], thus suggesting chickens as important reservoirs of Salmonella in Nigeria.The cause of the high prevalence rate in farms J and K is unknown due to limited data.
In this study, the predominant serovar in chicken was S. Virchow, which is one of the most common serovars in chicken [46,61,[63][64][65][66][67].In Europe, S. Virchow was listed as the seventh most common serovar isolated from broiler meat in 2007 [46].S. Virchow was observed in all farms expect G, with a high prevalence of 24% and 13% in farms J and K. Interestingly, S. Enteritidis was not isolated from chicken in this study.In contrast, S. Enteritidis is the most predominant serovar in Gallus Gallus flocks in the European Union, with a prevalence of 38% [46].This difference may be explained by differences in export patterns, production systems, climate, or feed.Recently, S. Hiduddify, a rare serovar, was found to be the predominant serovar in chickens in Maiduguri, Northern Nigeria [20], but was also not isolated in the fecal samples of chickens in this study.Northern Nigeria is generally warmer than the region of this study; moreover, the chickens studied by Raufu et al. were largely free range in contrast to the intensively reared chickens we studied, which this may suggest the presence of S. Hiduddify in the environment where chickens likely pick up the serovar from foodstuffs as they scavenge for foods in the environment.This hypothesis needs to be confirmed in free-range chickens in the south or by investigation of the environment in Maiduguri, Nigeria.We also isolated S. Onireke, a rare serovar in chickens from Ibadan, Nigeria.We speculate that those rare isolates found in chicken also originate from reptiles or the environment like the rare serovars obtained from invasive Salmonella in humans, but further research is needed to explore this hypothesis.
Although none of the serovars obtained from chicken fecal samples was isolated from the analysed human blood samples, those serovars still constitute potential serious health risks for humans in Ibadan, Nigeria.In Europe, S. Virchow and S. Derby are among the ten most frequent serovars causing human salmonellosis [46] and may be found in human stool samples from Ibadan, Nigeria, if investigated.A plausible explanation for why we could not link any of the serovars obtained from chickens to humans may be due to the analysis of human blood and chicken fecal samples in different years, suggesting time-based shifts in the prevalence of serovars.Analysis of blood samples from only humans could also be responsible because all serovars of NTS are not equally invasive.
The Salmonella isolates from chickens were more commonly resistant to tetracycline, sulfamethoxazole, nalidixic acid, and trimethoprim; this resistance may be attributed to indiscriminate use of antibiotics at recommended doses or at subtherapeutic doses as feed additives to promote growth, and as chemotherapeutic agents to control epizootics on the farms [68].The high level of resistance to nalidixic acid coupled with the high level of reduced susceptibility to ciprofloxacin is worrisome because fluoroquinolones are strategic in the treatment of invasive salmonellosis [60].The most resistant serovar from chicken was S. Virchow; this serovar is usually multiple resistant to antimicrobials [64,65,69].These observations call for regulation of antibiotic usage in Nigeria to stem the spread of resistance to antimicrobials.The finding of qnrS1 in S. Derby is another public concern because qnr genes mediate resistance to fluoroquinolones, which can compromise treatment with fluoroquinolones [60]; furthermore, qnr location on mobile genetic elements coupled with indiscriminate use of antibiotics in Nigeria will facilitate selection and potential spread to other serovars [70].

Conclusion
This study reports the relative importance of S. Enteritidis, S. Typhimurium, and S. Dublin associated with human bacteraemia in Ibadan, Nigeria, and that S. Typhi still constitutes a significant public health risk.This is the first time that S. Dublin has been reported as an important cause of invasive salmonellosis in sub-Saharan African countries.We also observed rarely isolated Salmonella serovars previously associated with reptiles causing invasive salmonellosis in humans.
As observed elsewhere in Nigeria, we can confirm the high prevalence rates of Salmonella spp.from chicken fecal samples, with S. Virchow as the predominant serovar.None of the serovars obtained from chicken fecal samples was isolated from the analyzed human blood samples; those serovars still constitute potential serious health risks for humans in Nigeria and should be controlled by targeted interventions.
A high level of reduced susceptibility to ciprofloxacin and resistance to nalidixic acid was observed in all serovars isolated from chickens.Additionally, we detected the plasmid mediated quinolone resistance gene, qnrS1, in S. Derby.

Table 3 .
et al. found that the main serovars obtained from children with bacteraemia in Kenya were S. Enteritidis (55%) and S. Typhimurium (33%) Number and percentages of isolates per serovars Frequency of and occurrence of Salmonella serovars from human blood samples by sites.Number (percentages) of antimicrobial resistance to the following antimicrobials

Table 4 .
Frequency of antimicrobial resistance in Salmonella isolates from human blood samples