Prevalence, sensitivity and specificity of antibodies against carbamylated proteins in a monocentric cohort of patients with rheumatoid arthritis and other autoimmune rheumatic diseases

Arbi Pecani
Cristiano Alessandri
Francesca Romana Spinelli
Roberta Priori
Valeria Riccieri
Manuela Di Franco
Fulvia Ceccarelli
Tania Colasanti
Monica Pendolino
Riccardo Mancini
Simona Truglia
Cristiana Barbati
Marta Vomero
Danilo Sabatinelli
Francesca Morello
Guido Valesini
Fabrizio Conti
Prevalence, sensitivity and specificity of antibodies against carbamylated proteins in a monocentric cohort of patients with rheumatoid arthritis and other autoimmune rheumatic diseases
10.1186/s13075-016-1173-0R E S E A R C H A R T I C L E Open Access
Background: Antibodies against carbamylated proteins (anti-CarP) have been recently identified in the sera of patients with rheumatoid arthritis (RA). The objective of the study was to evaluate the prevalence, sensitivity and specificity of anti-CarP compared to anti-citrullinated peptide antibodies (ACPA) and rheumatoid factor (RF), replicating the existing data in a large cohort of Italian patients with RA and extending the evaluation to other autoimmune rheumatic diseases (AIRDs). Methods: Serum samples (n = 607) from 309 patients with RA, 200 disease controls and 98 normal healthy subjects (NHS) were evaluated. Anti-CarP were detected using carbamylated fetal calf serum as the antigen. ACPAs were detected using second-generation ELISA and IgM RF was assessed as part of routine analysis. Results: Anti-CarP antibodies were detected in 117 patients with RA (34.4%), ACPA in 190 patients (61.4%) and RF in 202 patients (65.3%). Two (2.04%) of the NHS were positive for anti-CarP, one NHS (1.02%) was positive for ACPA and three NHS were positive for RF (3.06%). Among disease controls, anti-CarP antibodies were detected in 33 patients (16.5%), ACPA in 29 patients (14.5%) and RF in 64 patients (32%). In particular, 16.8% of patients with systemic lupus erythematosus and 31.1% of patients with Sjögren syndrome were positive for anti-CarP. The sensitivity of anti-CarP, ACPA and RF was 46.8%, 61.8% and 64.4%, respectively and specificity was 91.95%, 89.93% and 76.51%, respectively. Conclusions: The present study extends the knowledge of anti-CarP antibodies, confirming previous data on the diagnostic accuracy of anti-CarP in RA in a large cohort of Italian patients. Anti-CarP antibodies demonstrated relatively low sensitivity and slightly higher specificity compared to ACPA and RF. Even if predominantly present in RA, anti-CarP was detected in a variable percentage of patients with other autoimmune rheumatic diseases and their generation could be attributed to the inflammatory status; the clinical relevance of anti-CarP antibodies in these latter patients should be further determined.

Background

The discovery of autoantibodies in patients with RA facilitated the subgrouping of these patients for more accurate therapeutic management, resulting in more efficient disease control [1]. Beside the well-known rheumatoid factor (RF), anti-citrullinated protein antibodies (ACPA) have been reported to be a very useful diagnostic and prognostic marker of RA [2]. ACPA have remarkable sensitivity for this disease, with high predictive value for RA development and severity [2,3]. The importance of ACPA in RA was highlighted by the inclusion of ACPA status in the 2010 classification criteria for RA by allowing the division of patients with RA into two major subsets: ACPApositive and ACPA-negative [4].

Although ACPA have an important role in the diagnosis of RA, there is still a continuous demand for new biomarkers to further improve the early diagnosis of RA and especially its seronegative subgroup [5]. Recently, a new autoantibody system recognising antibodies against carbamylated proteins (anti-CarP) has been described [6] but has not yet been implemented for commercial use.

Initially, Shi et al. identified homocitrulline as the main aminoacid involved in the binding of autoantibodies in seronegative patients with RA [7]. Carbamylation is a chemical reaction mediated by cyanate that modifies lysine residues [5]. Normally the level of cyanate is in equilibrium with urea but specific conditions like inflammation can warp this equilibrium through a myeloperoxidasedependent mechanism [8,9]. This leads to the local increase of cyanate levels, thus empowering the degree of carbamylation [10].

Although the high resemblance in structure between citrulline and homocitrulline, inhibition and cohort studies have demonstrated that ACPA and anti-CarP are different and independent antibody subsets that do not cross-react with each other [4,5]. Unlike ACPA, the presence of anti-CarP has not been associated with HLA-shared epitope (SE) and/or smoking [11]. An interesting finding was the presence of anti-CarP in ACPAnegative patients with RA and their association with increased disease activity [12,13] and severe joint damage [13,14]. Moreover, anti-CarP have been detected in patients with arthralgia and their presence has been independently associated with the risk of developing RA [15]. In addition, anti-CarP are present in serum from patients with RA many years before the clinical appearance of the disease [14,16,17] and have also been identified in healthy first-degree relatives of patients with RA [18].

Recently, Shi et al. investigated the diagnostic performance of anti-CarP in RA in a large cohort of patients with early arthritis and demonstrated that these antibodies are predominantly detected in RA, but that a small percentage of patients with almost all forms of early arthritis were also anti-CarP-positive [19]. Considering the high prognostic and predictive value that anti-CarP have demonstrated in patients with RA, the aim of this study was to evaluate their prevalence, sensitivity and specificity in a large monocentric cohort of patients with RA compared to other autoimmune rheumatic diseases (AIRDs).

Methods

Patient populations

We evaluated a total of 607 frozen stored serum samples from 309 patients with established RA diagnosed according to the 2010 American College of Rheumatology (ACR)/ European League Against Rheumatism (EULAR) criteria, and 200 unselected patients with other diseases [systemic lupus erythematosus (SLE), Sjögren syndrome (SS), systemic sclerosis (SSc), and postmenopausal osteoporosis (OP)], who were not matched by gender or age with the RA group, and who were attending outpatient clinics at Sapienza University of Rome. Serum samples from 98 unselected normal healthy subjects (NHS) were also tested.

Anti-CarP antibody assays

Anti-CarP antibodies were detected by a modified solid phase "home-made" ELISA as described by Shi et al., with some modifications [20] using carbamylated fetal calf serum (FCS) as the antigen. In brief, Nunc Maxisorp plates (Thermo Scientific, Whaltam, MA, USA) were coated overnight at +4°C with non-modified FCS and Ca-FCS (10 μg/ ml in carbonate bicarbonate buffer). After washing the plates were blocked with phosphate-buffered saline (PBS) 1% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) for 6 h at +4°C. Subsequently, the wells were incubated with serum from patients, diluted 1/50 in PBS/ 0.05% tween/BSA 1% overnight at +4°C. After four washes the plates were incubated for 2 h at room temperature (RT) with goat polyclonal antihuman IgG alkaline phosphatase conjugated antibodies (Sigma-Aldrich, St. Louis, MO, USA) diluted at 1:1000 in PBS/0.05% tween/BSA 1%. After four washes a solution of paranitrophenyl phosphate tablets in ethanolamine was used for the enzyme reaction and the plates were read at a 405 nm wavelength after 30 minutes at RT. All assays were performed in duplicate and the absorbance of control wells (unmodified FCS) was subtracted to account for non-specific binding. The levels were determined in arbitrary units per millilitre (AU/ml) using a standard curve. The cutoff for anti-CarP antibody ELISA was established as the mean plus three times the standard deviation (SD) of the healthy control.

Anti-CCP and RF antibody assays ACPA were detected using a second-generation ELISA (anti-CCP) kit (Delta Biologicals, Italy) while IgM RF was determined as part of routine analysis by immunonephelometry (Behering, Marburg, Germany) according to the manufacturers' instructions.

Statistical analysis

The diagnostic performance of ACPA, RF and anti-CarP antibodies assays was determined by receiver operating characteristic (ROC) curve analysis, by plotting sensitivity (%) against 100-specificity (%) at different cutoff values. Diagnostic sensitivity was compared at cutoff levels corresponding to 95% specificity. Dunn's multiple comparison test was used to compare the levels of anti-CarP antibodies between the different diagnoses and the chi-square test was used to compare the percentage of the patients with other rheumatic conditions who were positive for the these antibodies. P values <0.05 were considered statistically significant. Data analysis was performed using SPSS version 20 (SPSS Inc., Chicago, IL, USA).

Results

We analysed serum from 309 patients with RA (mean age 55.4 ± 13.8 years), who had a mean disease duration of 107 ± 97.6 months; 238 (77%) were female, and 167 (54%) were smokers. Table 1 shows the characteristic of patients with RA and the control groups.

Anti-CarP, anti-CCP and RF in Patients with RA and controls

The overall prevalence of anti-CarP, RF, and anti-CCP in patients with RA, disease controls and healthy subjects is shown in Table 2.

The prevalence of anti-CarP antibodies was significantly higher in patients with RA compared to healthy controls (p = 0.00005) and non-RA disease (p < 0.00001 for SLE, SSc and OP) but not compared to SS (p = 0.05). Even when considering the disease controls as a whole group, the prevalence of anti-CarP antibodies was still significantly higher in patients with RA (p < 0.00001). Figure 1 shows the distribution of anti-CarP, ACPA and IgM-RF in patients with RA ( Fig. 1a) and non-RA disease (Fig. 1b); most of the patients with RA were positive for all three antibodies tested (27% of patients) or as combination of two antibodies. Conversely, among the non-RA disease controls only two patients with SLE had double positivity for anti-CarP and ACPA and other two were triple-positive and were diagnosed as having Rhupus; moreover, two patients with SS were triple-positive and one of them was diagnosed as having RA-associated SS. Interestingly, in the subset of seronegative patients with RA, 29% of these patients (25 out of 87) had anti-CarP antibodies ( Fig. 1c and d).

When evaluating the association between RA subsets according to antibody positivity and cigarette smoking there was an association with the presence and titre of ACPA (p = 0.001) but not with anti-CarP positivity or with RF ( Table 3). None of the autoantibodies correlated with disease activity as measured by disease activity score in 28 joints (DAS 28).

The AUC from ROC analysis (Fig. 2) was 0.678 (95% CI 0.636-0.720) for anti-CarP, 0.858 (95% CI 0.830-0.887) for ACPAs and 0.796 (95% CI 0.761-0.831) for RF. Among the three antibodies studied, anti-CarP antibodies had the highest specificity but relatively low sensitivity (91.9 and 46.8, respectively). Table 4 summarizes the diagnostic performance of anti-CarP compared to ACPA and RF.

Anti-CarP serum levels in patients with RA and in controls are shown in Fig. 3. When comparing the anti-CarP titre in positive patients we noticed that the levels were significantly higher in patients with RA compared to non-RA conditions (p < 0.05 for all conditions) (Fig. 3). Nevertheless, high levels of anti-CarP antibodies were detected even in the few patients with SLE and SS, although these levels were never as high as the levels of anti-CarP antibodies in the patients with RA (Fig. 3). Anti-CarP, ACPA and IgM-RF were not statistically different among the patients with non-RA disease (p > 0.05 for all).

Discussion

In recent years, the importance of early diagnosis and treatment toward more efficient control of the disease,     less joint damage and a better outcome has been clearly stated. For this reason, research has mostly focused on identifying biomarkers and specific laboratory tests to be applied in the early detection of RA [1]. The present study analyses prevalence, sensitivity and specificity of anti-CarP antibodies, replicating previously published data in a large, single-centre cohort of Italian patients with RA compared to healthy controls and patients with other AIRDs. For the first time we investigated in a single study the positivity of anti-CarP, even including patients with different systemic AIRDs, which are known to be associated with the production of several autoantibodies. In this study we confirmed the data on anti-CarP showing the presence of these antibodies in a significant number of patients with RA. Actually, 15 patients were single-positive for ACPA and 26 were single-positive for anti-CarP, confirming the lack of cross-reactivity between anti-citrullinated and anti-homocitrullinated proteins as previously described [6]. Isolated anti-CarP positivity was detected in about one third of patients with RA who were seronegative for ACPA and RF.

The major advantage of simultaneous or alternative detection of a wide range of biomarkers consists in a  Anti-CarP antibodies against carbamylated proteins, ACPA anti-citrullinated protein antibodies, RF rheumatoid factor, LR likelihood ratio, PPV positive predictive value, NPV negative predictive value much detailed classification of RA subtypes, increasing the sensitivity of RA diagnosis [2,5]. As suggested by the results of previous research [6,7], in this study we were able to identify different immunological RA subsets based on the presence and/or absence of the three autoantibodies studied (anti-CarP, ACPA and RF) opening a new window of opportunity to more precise disease classification and treatment. When comparing the findings of the present study with previous results, there is a difference in the percentage of triple positivity for anti-CarP, ACPA and RF: indeed, the percentage of triplepositive patients is lower among patients in the Norfolk Arthritis Register and higher in the early arthritis Leiden cohort compared to the present cohort of Italian patients with RA [12,19]; this discrepancy could result from the different genetic background of the populations studied. Moreover, unlike the results of a longitudinal study, reporting higher disease activity over time in anti-CarP-positive patients [12], the findings of our crosssectional analysis agree with the lack of association with disease activity reported by others [13]. Unlike citrullination -that is an enzimatically mediated reaction -carbamylation is a chemical reaction that occurs in the presence of a reactive metabolite (cyanate) [6]. Theoretically, every protein can be carbamylated in vivo but the probability of a protein undergoing such modification depends on various parameters such as the number and accessibility of lysine and arginine amino groups and the protein lifespan [7]. As carbamylation is an almost irreversible reaction, it is more likeky to affect long-lived proteins as they may have acquired homocitrulline residues over time [7]. Taken together, these data suggest that even the antibodies produced by these posttranslational modification reactions may differ in terms of sensitivity and specificity. Thus, in the present study, we compared the diagnostic performance of anti-CarP antibodies, ACPAs and RF in a large monocentric cohort of patients with RA compared to disease and healthy controls with different AIRDs.

As already mentioned, anti-CarP antibodies were present in a significant percentage in seronegative and seropositive patients with RA. Beside patients with RA, anti-CarP antibodies were present in a small number of patients with SLE and SS; less than one third of these patients also displayed ACPA positivity. These results can be partially attributed to erosive joint disease described in a small percentage of patients with SLE [21]; besides, carbamylation can be enhanced in patients with kidney involvement or in those with active inflammatory status [8]. Similarly, a clear link between antibody production and the degree of inflammation has been shown in patients with SS, as the activity of circulating myeloperoxidase (MPO), which catalyzes the production of cyanate from thiocyanate, seems to be significantly increased [20]. Two studies investigated anti-CarP in patients with SLE and with SS [20,21]. The detection of a subset of anti-CarP-positive subjects among patients with SLE or SS and joint involvement suggests the possibility of that anti-CarP might serve as a serological marker for erosive arthritis in AIRDs other than RA. However, the clinical and prognostic relevance of these autoantibodies in patients with non-RA needs to be further elucidated.

In diagnostic performance, anti-CarP antibodies had slightly higher specificity but relatively lower sensitivity when compared to ACPA and IgM-RF. On the other hand, the AUC for anti-CarP antibodies in the whole population was 0.678, whereas in the seronegative group it was only 0.565. These data suggest an additional role of anti-CarP positivity in the diagnosis of RA, in agreement with previous studies reporting the presence and prognostic value of anti-CarP antibodies in the RA seronegative subgroup, and correlation with joint destruction [7,12,13,15]. The diagnosis of RA is mostly clinical, and it is supported by serological data. Nonetheless, as we are moving toward the identification of RA in its early phases, biomarkers could help in stratifying subjects at risk of developing overt disease even in pre-clinical phases [4,5].

Conclusions

In conclusion, the results of the present study extend the knowledge on anti-CarP providing updated data on prevalence, sensitivity and specificity on a large cohort of Italian patients. Moreover, we confirm that anti-CarP antibodies and ACPA are not always present simultaneously. The positivity of these antibodies in patients with non-RA autoimmune rheumatic disease can be attributed to the inflammatory status and should be addressed in further studies in order to better understand their role in autoimmune diseases.

Fig. 1
1Distribution of antibodies against carbamylated proteins (anti-CarP), anti-citrullinated protein antibodies (ACPA) and rheumatoid factor (RF) in patients with rheumatoid arthritis (RA) and disease controls. a-b Venn diagram showing the relationship between anti-CarP antibodies, ACPA and IgM RF in patients with RA (a) and other conditions (non-RA) (b). c Anti-CarP positivity in the whole RA population and double-negative patients with RA. d Distribution of single, double and triple positivity for anti-CarP, ACPA and RF in patients with RA

CarP (−)/ACPA (+)/RF (CarP (−)/ACPA (−)/RF (+) 26 14 (53.8%) 12 (46.2%) −4.2-0.2 ns Anti-CarP (+)/ACPA (+)/RF (−) 4 2 (50%) 2 (50%) −2.2-2.2 ns Anti-CarP (+)/ACPA (−)/RF (+) 5 3 (60%) 2 (40%) −3.2-1.2 ns RA rheumatoid arthritis, Anti-CarP antibodies against carbamylated proteins, ACPA anti-citrullinated protein antibodies, RF rheumatoid factor, ns not significant

Fig. 2
2Receiver operating characteristic curve for antibodies against carbamylated proteins (anti-CarP), anti-citrullinated protein antibodies (ACPA) and rheumatoid factor (RF)

Fig. 3
3Antibodies against carbamylated proteins (anti-CarP) in serum from patients and controls. Horizontal line corresponds to cutoff value: *p < 0.0005 for rheumatoid arthritis (RA) vs normal healthy subjects (NHS) and patients with osteoporosis (OP); **p = 0.03 for RA vs systemic lupus erythematosus (SLE); ***p = 0.04 for RA vs Sjögren syndrome (SS); ****p = 0.004 RA vs systemic sclerosis (SSc); § p = 0.00001 for SLE vs NHS; § § p = 0.002 for SLE vs SSc; § § § p = 0.05 for SLE vs OP; # p = 0.00001 for SS vs NHS and SS vs SSc; † p = 0.03 for SSc vs NHS; † † p = 0.004 for SSc vs OP

Abbreviations
ACPAs: anti-citrullinated protein antibodies; ACR: American College of Rheumatology; AIRDs: autoimmune rheumatic diseases; anti-CarP: antibodies against carbamylated proteins; anti-CCP: anti-cyclic citrullinated peptides; AU: arbitrary units; AUC: area under the curve; BSA: bovine serum albumin; DAS28: disease activity score in 28 joints; ELISA: enzyme-linked immunosorbent assay; EULAR: European League Against Rheumatism; FCS: fetal calf serum; Ig: immunoglobulin; LR: likelihood ratio; NHS: normal healthy subjects; NPV: negative predictive value; OP: osteoporosis; PBS: phosphate-buffered saline; PPV: positive predictive value; RA: rheumatoid arthritis; RF: rheumatoid factor; ROC: receiver operating characteristic; RT: room temperature; SD: standard deviation; SE: shared epitope; SLE: systemic lupus erythematosus; SS: Sjögren syndrome; SSc: systemic sclerosis

Table 1
1Characteristic of study participants RA rheumatoid arthritis, SLE systemic lupus erythematosus, SS Sjögren syndrome, SSc systemic sclerosis, OP osteoporosis, HS healthy subjectsDisease
(number of patients)

Age (years)
(mean + SD)

Female
n, (%)

Smokers
n, (%)

RA (309)
55.4 + 13.8
238 (77)
167 (54)

SLE (83)
42.8 + 11.3
57 (68.6)
34 (40.9)

SSC (51)
50.4 + 12.1
51 (100)
10 (19.6)

SS (45)
53.3 + 13.1
30 (66.6)
26 (48.1)

OP (21)
66.1 + 7.2
21 (100)
2 (9.5)

HS (98)
53.1 + 10.4
73 (74.5)
43 (43.9)

Table 2
2Prevalence of anti-CarP, ACPA and RF in patients with RA, disease controls and healthy subjects Anti-CarP anti-carbamylated proteins antibodies, ACPA anti-citrullinated peptides antibodies, RF rheumatoid factor, RA rheumatoid arthritis, NHS normal healthy subjects, SLE systemic lupus erythematosus, SS Sjögren syndrome, SSc systemic sclerosis, OP osteoporosisDisease
(number of patients)

Anti-CarP positive,
n, (%)

ACPA positive,
n, (%)

RF positive,
n, (%)

RA (309)
117 (34.4)
190 (61.4)
202 (65.3)

NHS (98)
2 (2.04)
1 (1.02)
3 (3.06)

SLE (83)
14 (16.8)
15 (18.07)
19 (22.8)

SS (45)
14 (31.1)
11 (24.4)
23 (51.1)

SSc (51)
3 (5.8)
1 (1.9)
18 (35.2)

OP (21)
2 (9.5)
2 (9.5)
4 (19.04)

Table 3
3Association between smoking and different RA subsets according to the presence/absence of anti-CarP antibodies, ACPA and RFSmoking

Disease subset
Patients, n
Never smoked, n (%)
Ever smoked, n (%)
95% CI
P

Table 4
4Diagnostic performance of anti-CarP antibodies, ACPA and RFSensitivity (95% CI) Specificity (95% CI) LR
PPV NPV

Anti-CarP 46.8 (41.50-52.54) 91.95 (82.25-94.77) 4.581 88% 60%

ACPA
61.8 (56.14-67.25) 89.93 (85.94-93.10) 6.14 85% 62%

RF
64.4 (58.78-69.74) 76.51 (71.28-81.21) 2.742 80% 69%

AcknowledgementsThis work has been supported by Fondazione Umberto Di Mario ONLUS. We thank Giuliana Giuliani and Stefania Mieli for their technical support.FundingNot applicable.Availability of data and materialsDatasets analyzed during the current study are available from the corresponding author on reasonable request.Authors' contributions AP designed the study, collected patients' samples and tested the antibodies, analysed and interpreted the data and drafted the manuscript. CA participated in the design of the study, analysed and interpreted the data and critically revised the manuscript. FRS collected samples and data from patients with systemic lupus erythematosus, analysed and interpreted the data and was a major contributor in writing the manuscript. RP enrolled patients for the study and collected clinical data from patients with Sjogren Syndrome. VR enrolled patients for the study and collected clinical data from patients with scleroderma. MDF enrolled patients for the study and collected clinical data from patients with rheumatoid arthritis. FuC enrolled patients with systemic lupus erythematosus for the study and collected clinical data. TC developed and carried out the immunoassay for anti-CarP antibodies and analysed and interpreted the data. MP collected samples from patients with osteoporosis and healthy controls and tested the anti-CarP antibodies. RM conceived the study and developed the immunoassay for anti-CarP antibodies. ST enrolled patients for the study, collected clinical data and performed the statistical analysis. CB performed the immunoassay for RF and ACPA and analysed the data. MV performed the immunoassay for RF and ACPA. DS performed the immunoassay for RF and ACPA. FM enrolled patients for the study and collected clinical data from patients with rheumatoid arthritis. GV designed the study, analysed and interpreted the data and critically revised the manuscript. FaC enrolled patients for the study, and collected clinical data and critically revised the manuscript. All authors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.Consent for publicationNot applicable.Ethics approval and consent to participate Written informed consent was obtained from all the patients and control subject enrolled in the study. The study was approved by the local Ethical Committee (Comitato Etico Sapienza Università di Roma -Policlinico Umberto I).Received: 28 July 2016 Accepted: 7 November 2016• We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal Submit your next manuscript to BioMed Central and we will help you at every step:
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Anti-Carbamylated protein antibodies in unaffected first-degree relatives of rheumatoid patients: lack of correlation with anti-cyclic citrullinated protein antibodies and rheumatoid factor. C Alessandri, I Bartosiewicz, M Pendolino, R Mancini, T Colasanti, A Pecani, Clin Exp Rheumatol. 33Alessandri C, Bartosiewicz I, Pendolino M, Mancini R, Colasanti T, Pecani A, et al. Anti-Carbamylated protein antibodies in unaffected first-degree relatives of rheumatoid patients: lack of correlation with anti-cyclic citrullinated protein antibodies and rheumatoid factor. Clin Exp Rheumatol. 2015;33:824-30.

The specificity of anti-carbamylated protein antibodies for rheumatoid arthritis in a setting of early arthritis. J Shi, H W Van Steenbergen, J A Van Nies, E W Levarht, T W Huizinga, A H Van Der Helm-Van Mil, Arthritis Res Ther. 17339Shi J, van Steenbergen HW, van Nies JA, Levarht EW, Huizinga TW, van der Helm-van Mil AH, et al. The specificity of anti-carbamylated protein antibodies for rheumatoid arthritis in a setting of early arthritis. Arthritis Res Ther. 2015;17:339.

Anti-Carbamylated Protein Antibodies Identify Systemic Lupus Erythematosus Patients with Erosive Arthritis: Analysis of a Regional. A Kastbom, P Wallin, M Ziegelasch, T Skogh, L Trouw, C Sjöwall, Swedish Register [abstractKastbom A, Wallin P, Ziegelasch M, Skogh T, Trouw L, Sjöwall C. Anti- Carbamylated Protein Antibodies Identify Systemic Lupus Erythematosus Patients with Erosive Arthritis: Analysis of a Regional Swedish Register [abstract].

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Antibodies against carbamylated proteins are present in primary Sjogren's syndrome and are associated with disease severity. B Berqum, C Koro, N Delaleu, M Solheim, A Hellward, V Binder, Ann Rheum Dis. 75Berqum B, Koro C, Delaleu N, Solheim M, Hellward A, Binder V, et al. Antibodies against carbamylated proteins are present in primary Sjogren's syndrome and are associated with disease severity. Ann Rheum Dis. 2016;75:1494-500.


Salmonella and Escherichia coli contamination of poultry meat from a processing plant and retail markets in Ibadan, Oyo State, Nigeria

Gladys Taiwo Adeyanju
Olayinka Ishola
Salmonella and Escherichia coli contamination of poultry meat from a processing plant and retail markets in Ibadan, Oyo State, Nigeria
R E S E A R C H Open AccessPoultry meatSalmonella sppEscherichia coliAntibioticsResistance
Salmonella spp and Escherichia coli are the two most important food-borne pathogens of public health interest incriminated in poultry meat worldwide. This study is to access their levels in frozen poultry meat obtained in Ibadan, Oyo State and compare those obtained from a commercial Nigerian-registered poultry company having a broiler-processing plant, Sayed Farms Ltd(R), with that obtained from retail stores. These retail stores source their products as illegal imports from neighboring Benin Republic or Togo because of a ban imposed by Government policy in Nigeria since July 2002 (USDA, GAIN report #NI2025:1-6, 2002). Microbiological Standards and Guidelines by USDA (National Agricultural library) (USDA 2011) and NCCLS guidelines (from Global Salm-Surv, 2003)  were used during the research work. The study was approved by the Ethical Research Review Board (ERRB, Research Management Office 2011), University of Ibadan, Nigeria. A total of one hundred and fifty-two (152) frozen poultry meat samples comprising ninety-nine retail poultry (53 chicken and 46 turkey) and 53 chicken from the processing plant were accessed. ISO Standards catalogue 07.100.30 (2011) was used in accessing the levels of Salmonella, Escherichia coli, Enterobacteriaceae counts and Aerobic plate count. ISO 6579: 2002 was used for Salmonella isolation and ISO-16654:2001 for Escherichia coli isolation. There was a higher level of Aerobic plate counts and Enterobacteriaceae counts in frozen retail poultry meat than from the processing plant. Salmonella contamination from the ninety-nine poultry samples (53 chicken and 46 turkey) obtained from retail markets was at 33% [chicken 32.1% (17/53) and turkey 34.8% (16/46)] while Escherichia coli at 43.4% [chicken 47.2% (25/53) and turkey 39.1% (18/46)]. From the processing plant, twelve (12) Salmonella isolates were obtained and prevalence rate calculated as 22.6% while three (3) Escherichia coli isolates at 5.7% was obtained. Antibiotic sensitivity for isolates using eight different Gram-negative antibiotics showed different resistance patterns. Nitrofurantion and augmentin showed a decrease in their sensitivity to isolates than they normally should. Salmonella enterica spp. showed 93% resistance to tetracycline and 100% resistance to augmentin and amoxicillin, while Escherichia coli showed 100% resistance to augmentin and amoxicillin.

Introduction

Turkey, spent layers and majorly, broilers serve as sources of poultry meat which has become a product accepted and consumed worldwide as there is an absence of cultural or religious obstacles associated with their use as food and Nigeria is no exception (Alabi and Alabi 2009).

Major exports of poultry to West Africa comes from the European Union (EU), the exports of which has increased from 12500 tons in 1996 to 86000 tons in 2003, mainly to Benin, followed by Ghana, Nigeria, Senegal, Togo and Ivory Coast. Imports into Nigeria has been banned since July 2002 in order to boost local production, but it continues although undocumented (USDA, GAIN report 2002). The European community (EC) was banned from the use of antimicrobials as growth promoters in poultry faming in January 2006 because of the risks of increased resistance (Miranda et al. 2008) but demand and imports of poultry meat into Nigeria has increased over the years. Nigeria annually produces an average 11829 tons of chicken, spending about 8 million US dollars to import chicken meat in 2005 alone (Alabi and Alabi 2009). Poultry meat (chicken and turkey) is readily available for sale in retail markets areas such as is obtainable in Ibadan, Oyo state, Nigeria. These imported poultry are kept in freezers but power supply is not constant and is augmented by the use of fuel-powered generating sets. It should be noted however that ensuring consumer health concerns the greater involvement of the health sector, development of Codex standards, guidelines and incorporation of the work of the Commission into the national legislation to promote food safety and fair trading practices as should be reflected in the priorities of the Codex Alimentarius Commission of any country especially in the developing countries.

During the slaughter of poultry birds, there can be fecal contamination of the carcasses from the gut of these birds which means bacteria present in the spilled gut content is passed on as contaminants. Of importance is the coliforms especially Escherichia coli and Salmonella. Collibacillosis and Salmonellosis have been described as the leading causes of food-borne illnesses worldwide (Panisello et al. 2000), therefore, it becomes important that ensuring consumer health concerns the greater involvement of the health sector.

Salmonella is of an increasing public health concern because they are the most incriminated pathogenic microorganisms of bacterial food poisoning especially present in poultry meat, with infection being through the handling of raw poultry carcasses and products, together with the consumption of undercooked poultry meat (Panisello et al. 2000).

The modernization of chicken farms and globalization of the bird breeding trade also have played a role in infection (Velge et al. 2005), with several serotypes being isolated from retail poultry products from many years back in various parts of the world (Rindhe et al. 2008). Prevalence of Salmonella in poultry meat using both traditional and conventional methods has been reported worldwide from retail outlets, retail markets and processing plants. It has been reported to be as low as 1.56% from a Morocco poultry processing plant (Cohen et al. 2007) and as high as 20% from a poultry processing plant in USA (Russell 2009). In retail markets, prevalence was reported in broilers at 10.60% in Croatian market (Kozačinski et al. 2006), 31% in India (Dahal 2007), 35.5% in Mexico (Miranda et al. 2009) and 5.92% in Saudi Arabia (Moussa et al. 2010). In Nigeria, several rates have been reported with 11.1% prevalence in Calabar metropolis (Ukut et al. 2010) and 2% in Osogbo (Adesiji et al. 2011). Resistance to ampicillin appears to be the most common in Nigeria, followed by trimethoprimsulphamethozazole, streptomycin, cephalexin, gentamycin (Enabulele et al. 2010) and more than 90% resistance to tetracycline (Sakaridis et al. 2011).

E. coli, a natural inhabitant of the intestinal tracts of humans and warm-blooded animals, is used as an indicator bacterium because it acquires antimicrobial resistance faster than other conventional bacteria (Miranda et al. 2008). Its presence therefore reliably reflects faecal contamination, indicating a possible contamination by enteric pathogens. Many different types of foods are sources of the bacterium and have been identified as a potential source of Shiga Toxin-producing Escherichia coli (STEC) for which such raw or undercooked foodstuffs get contaminated either during primary production (e.g. slaughtering) or further processing and handling (e.g. cross contamination during processing, human-to-food contamination via food handlers). E. coli has been isolated worldwide from poultry meat (Canton et al. 2008;Adesiji et al. 2011), probably due to the increased usage of antimicrobials (Miranda et al. 2008). Percentage prevalence in poultry meat has been variable depending on method and media used in its isolation. 19% prevalence was observed in South Africa (Dahal 2007), 48.4% in Morocco (Cohen et al. 2007) and as high as 98% in India (Saikia and Joshi 2010). In Nigeria, 16% has been isolated in Osogbo (Adesiji et al. 2011) and 11.1% in Calabar metropolis (Ukut et al. 2010). Enteroaggregative E. coli (EAEC) and Enterotoxigenic Fluoroquinolone resistant E. coli strains often show resistance to other drugs such as ampicilin, tetracycline, chloramphenicol, trimethoprin, sulphamethoxazole and Gentamicin (Komp et al. 2003), with a significant increase in fluoroquinolones resistant E. coli in many countries over the last few decades (Viroy et al. 2005). In Nigeria, the same results of E. coli resistance to quinolones and the other drugs have also been identified from poultry and beef sources (Olatoye 2010;Adesiji et al. 2011;Adetunji and Isola 2011). It has therefore been suggested that the resistance to the penicillin group of drugs, tetracycline, chloramphenicol and gentamicin may have been precipitated by the resistance to the quinolones (Komp et al. 2003).

Materials and methods

Sample collection

The study was carried out in Ibadan metropolis comprising Ibadan North, Ibadan North-West, Ibadan North-East, Ibadan South-West and Ibadan South-East. Sayed Farm Ltd®, is a commercial poultry company located in Ibadan South-East, registered in Nigeria, having a hatchery and a processing plant. The hatchery is involved in the hatching of both meat birds (broilers) and egg-layer birds, while the processing plant is involved in processing table-size broilers which are frozen before been sold.

A total of one-hundred and six chicken were sampled (fifty-three samples from retail markets and fifty-three samples from the processing plant) using Thrusfield method (Thrusfield 2005) while forty-six turkey were randomly sampled from the retail market. Research work was carried out within the months of April to July 2011.

Samples obtained were placed in sample-collection bags, properly labeled, stored in a cold box and taken to the laboratory for immediate analysis. However, no reference strains were used as quality control during the microbiological detection and isolation. One-way ANOVA and multiple comparisons were the statistical tools used while disk diffusion method was used for antibiotic sensitivity.

Microbiological Standards and Guidelines by USDA, (National Agricultural library) (USDA 2011) and NCCLS guidelines (from Global Salm-Surv, 2003) were used during the research work. The study was approved by the Ethical Research Review Board (ERRB), University of Ibadan, Nigeria (ERRB, 2011).

Microbiological procedure Aerobic plate count (APC) and Enterobacteriaceae counts (ENT)

25 g of test sample (meat) was weighed and blended in a stomacher machine for 2 minutes. A gram of the sample was weighed out and homogenised in 9mls buffered peptone water (LabM, UK) to give a dilution of 1:10. A six-fold serial dilution was then prepared. 0.1 ml of dilutions 10 −6 and 10 −5 for every sample was respectively plated on plate count agar (PCA) (Biomark, India) for aerobic plate count determination and on McConkey agar (MCA) (LabM, UK) for Enterobacteriaceae (enteric bacteria) counts. The PCA and MCA were both incubated overnight (18-24 hours) at 37°C.

Distinct colonies on PCA and MCA were counted using a digital colony counting chamber and recorded in colony forming units per gram (cfu/g) of meat sampled using the formula:

cfu=g ¼ level of dilution plated  number of colonies counted=volume plated:

These were further expressed in mean colony forming units per gram (mean cfu/g) and converted to log 10 base values. Statistically, SPSS statistical tool using One-way ANOVA and multiple comparisons were calculated.

Salmonella detection and isolation

ISO-6579: 2002 food microbiology procedure employing the horizontal method for the detection of Salmonella from food and animal feeding stuffs was used (ISO Standards catalogue 07.100.30; WHO 2010).

a. Non-selective pre-enrichment: 25 g of meat sample was collected under sterile conditions and transported to the laboratory in a maintained cold chain. It was blended in a stomacher machine for 2 minutes and a gram of the sample was weighed out and homogenised in 9 mls buffered peptone water (LabM, UK) in a test-tube to give a dilution of 1:10. Test-tubes were corked properly, labelled and incubated overnight (18-20 hours) at 37°C. b. Selective enrichment: Rappaport-Vassiliadis soya peptone (RVS) (Oxoid, England) was prepared. On cooling, 10mls was dispensed into test-tubes. 0.1 ml of the pre-enrichment was taken using a sterile pipette and transferred into the test-tube containing the Rappaport-Vassiliadis soya peptone broth. Test-tubes were covered using a cork, labelled and incubated overnight (18-24 hours) at 41.5°C ± 0.5°C. c. Selective agar plating: A 10 μl wire loop was used to pick a loop-full volume from the RVS broth and inoculated unto already prepared and solidified Brilliant green agar (BGA) (LabM, UK). Plates were spread out, labelled, inverted and incubated at 37°C overnight (18-24 hours). d. Sub-cultivation: suspect colonies which caused the colour of the medium to change from yellow to red/ pink were sub-cultivated unto nutrient agar (LabM, UK) and incubated overnight (18-24 hours) at 37°C.

Serotyping was carried out to determine incriminated specie for Salmonella using Polyvalent O Antisera. Two seperate loopful of normal saline was placed on a clean glass slide after which a clean wire loop was used to take a small part of suspect colony grown overnight on nutrient agar and placed on both drops of the normal saline and thoroughly mixed. A loopful of the antisera was placed on one of the bacteria suspension while normal saline was placed on the other (control). Both bacteria suspensions were mixed using a sterile wire loop and gently tilted to observe for agglutination. Positive Salmonella spp. with somatic antigen is indicative of agglutination reaction.

Escherichia coli isolation

ISO-16654:2001 food microbiology procedure of the horizontal method for the detection of Escherichia coli O157 from food and animal feeding stuffs was employed in this study (ISO Standards catalogue 07.100.30; WHO 2010). a. Enrichment: 25 g of meat sample was collected under sterile conditions and transported to the laboratory in a maintained cold chain. It was blended in a stomacher machine for 2 minutes and a gram of the sample was weighed out and homogenised in 9mls buffered peptone water (LabM, UK) in a test-tube to give a dilution of 1:10. Serial six-fold dilution in peptone water was prepared (10 −1 to 10 −6 ) for test samples. 0.1 ml of dilutions 10 −6 and 10 −5 for every sample was respectively plated on the MCA and spread out using glass spreader. Plates were labelled, inverted and incubated overnight (18-24 hours) at 37°C.

Antibiotic sensitivity test for isolates

Disk diffusion antibiotic sensitivity test was used in determining sensitivity of the Salmonella and Escherichia coli Gram-negative bacteria isolates obtained to various antibiotics at different micrograms. Augmentin (30 μg), Ofloxacin (5 μg), Gentamicin (10 μg), Nalidixic acid (30 μg), Nitrofurantoin (200 μg), Cotrimaxazole (25 μg) and Tetracycline (25 μg) were used to determine sensitivity pattern.

Results

Aerobic plate count and Enterobacteriaceae counts of frozen poultry Aerobic plate counts and Enterobacteriaceae counts in chicken

Mean (cfu/g) ± SD aerobic plate count (APC) for all the LGA's was calculated as 7.497 ± 0.94. However, there was no significant difference when one local government was compared with another using ANOVA. The only significant difference noted using LSD was between Ibadan South-East and Ibadan North-west at p value of 0.05 and between Ibadan South-West and Ibadan South-East at p value of 0.006 using multiple comparisons. Using ANOVA and multiple comparisons, no significant differences in ENT counts was observed when the means were calculated individually for each LGA and when they were compared one to another. Mean (cfu/g) ± SD Enterobacteriaceae counts for all the LGA's was calculated as 6.76 ± 1.93. Table 1 also shows logarithm mean counts in cfu/g. No significant difference was observed when the mean aerobic plate count and mean enterobacteriaceae counts in log 10 cfu/g of chicken from one market was compared with one another at p < 0.05 with 5% level of error. The level of bacterial contamination from one local government area to another showed only slight differences as reflected in the bar charts drawn in Figure 1. Mean APC was found to be higher than ENT counts interpreted as higher rates of aerobic bacteria contaminants on chicken.

Aerobic plate count and Enterobacteriaceae counts in turkey

Using ANOVA and multiple comparisons, no significant differences was observed when the mean total aerobic plate counts of turkey were calculated individually for each LGA and when they were compared one to another. Mean (cfu/g) ± SD aerobic plate count (APC) for all the LGA's was calculated as 7.42 ± 1.00. No significant differences was observed when the mean Enterobacteriaceae counts of turkey were calculated individually for each LGA and when they were compared one to another using ANOVA and multiple comparisons. Mean (cfu/g) ± SD Enterobacteriaceae counts for all the LGA's was calculated as 6.74 ± 2.00. Table 2 shows differences obtained amongst LGA's.

No significant difference was observed when the mean aerobic plate count and mean enterobacteriaceae counts in log 10 cfu/g of turkey from one market was compared to other market at p < 0.05 with only slight differences illustrated in their means as shown in the bar charts. APC in turkey is however higher than that obtained from Enterobacteriaceae counts, meaning that a higher contamination by aerobic bacteria was seen in turkey than enteric bacteria. Differences are illustrated in charts as seen in Figure 2. Figure 3 shows APC and ENT distribution obtained from retail poultry meat. Table 3 shows a representation and comparism between Aerobic plate count and Enterobacteriaceae counts obtained in chicken from retail market and processing plant which is graphically represented in Figure 4.

Extent of Salmonella and Escherichia coli contamination

From the one-hundred and six (106) chicken sampled comprising equal numbers of fifty-three from both retail market and processing plant, a total of twenty-nine (29) Salmonella spp. isolates, seventeen (17) from retail markets and twelve (12) from the processing plant were obtained while for Escherichia coli a total of twenty-eight (28) comprising twenty-five (25) from the retail market and three (3) from the processing plant were obtained. Percentages of isolates are shown in Figure 5.

Retail market

From the ninety-nine (99) poultry meat sampled (53 chicken and 46 turkey), thirty-three (33) Salmonella isolates (17 from chicken and 16 from turkey) were obtained at prevalence of 33% while a total of forty-three (43) Escherichia coli isolates (25 from chicken and 18 from turkey) were obtained at a prevalence of 43.4%.

Processing plant

From the 53 chicken samples obtained from the processing plant, twelve (12) Salmonella spp. isolates were obtained with prevalence of 22.6% while three (3) Escherichia coli isolates were obtained with prevalence of 5.7%. Antibiotic sensitivity test carried out on both Salmonella and Escherichia coli isolates shows the pattern of sensitivity represented in Tables 4 and 5 respectively.

Discussion

Aerobic plate count and Enterobacteriaceae counts of frozen poultry Retail market ENT counts were observed to be higher than APC for both chicken and turkey meat. This is because the agar used in determining APC is sensitive to all aerobic bacteria thereby allowing a wide range of bacteria to grow. MCA used in determining ENT is however limited in the range of bacteria that it allows its growth as it is only selective for enteric bacteria such as Escherichia, Shigella, Salmonella, Edwardsiella, Citrobacter, Yersinia, Klebsiella, Enterobacter, Serratia, Proteus, Morganella and Providencia as noted by Warren (2007). Slight differences were observed when comparing chicken with turkey meat. It however goes to say the samples from which these growths were obtained are contaminated at levels exceeding limits by food regulatory bodies, a reflection of the condition of the whole carcass meant for human consumption.

Processing plant

Mean aerobic plate count was calculated as 4.18 cfu/g which is less than 6.7 cfu/g obtained from a processing plant in Morocco (Cohen et al. 2007). Significant difference was observed in aerobic plate counts and enterobacteriaceae counts of chicken between processing plant and retail market at 0.006. Mean cfu/g ± SD of 3.99 ± 2.53 was obtained from processing plant. Although there were differences in Aerobic plate count and Enterobacteriaceae counts obtained between processing plant and retail market, where APC were noted to be higher than ENT counts, counts are reflection of the general sanitary condition of the carcasses and basically at high levels than permitted on such food.  LGA = Local Government Area, n = sample numbers collected, SD = standard deviation, cfu = colony forming unit, g = gram.

Salmonella and Escherichia coli Retail markets

Salmonella in more than 25 g of poultry meat is considered unsafe for human consumption. From the work understudied, Salmonella at levels higher than the recommended limits was obtained from both chicken and turkey. 33.3% (33) of poultry meat (chicken and turkey) from the ninety-nine samples obtained were contaminated with Salmonella. This percentage is higher than the 11.1% obtained in Calabar metropolis (Ukut et al. 2010) and the 2% from Osogbo (Adesiji et al. 2011) all from Nigeria. However, levels are indicative of the fact that if proper cooking is not done, Salmonella food-borne infection is most likely to occur. E. coli is recommended to be totally absent from poultry meat before such can be considered fit for human consumption. Levels obtained from work are reflections of the high rates obtained from markets. 43.4% (43/99) obtained from work is very high in comparison to 11.1% and 16% from Osogbo (Adesiji et al. 2011) and Calabar metropolis (Ukut et al. 2010) respectively. 11.3% prevalence reported from neighboring Cameron (Nzouankeu et al. 2010) is however lower than that from Nigeria even though we both have the same source of imports of these poultry meat. The rate of E. coli obtained is indicative that poultry meats obtained from sourced areas were unfit for human consumption in accordance with criterion of recommended limits by foreign food agencies. Poultry meat obtained from these markets should therefore be properly cooked to denature toxin produced by the organism as well as the organism such that consumption will not pose health-risks to human population.

Processing plant

The processing plant had a prevalence rate of 22.6% from chicken meat when compared to 1.56% from a processing plant in Morocco (Cohen et al. 2007) and 20% in USA processing plant (Russell 2009). Albeit, Salmonella contamination in poultry products from the processing plant is primarily due to cross contamination by physical contact during carcass processing such as improper cleaning and disinfection of processing lines, improper chilling and storage temperature, poor worker hygiene and infestation with rodents and insects.

The prevalence of Escherichia coli up to 94% was detected in chicken meat samples in the Netherlands which is quite alarming (HCV New Drug Research 2011) when compared to 5.7% obtained from study. These poultry products get contaminated with Escherichia coli along   processing line when intestinal contents accidentally spills on the meat been processed or due to contaminated equipments/storage/transporting facilities.

Antibiotic sensitivity tests Salmonella antibiotic sensitivity

The development of quinolone-resistant Salmonella resistant strains, especially those of poultry meat origin (Miranda et al. 2009), is gradually leading to an epidermic (White et al. 2001;Velge et al. 2005). Nalidixic acid is able to develop resistance quite rapidly. Several studies have also shown that resistance to nalidixic acid and decreased susceptibility to Fluoroquinolones have increased among Salmonella spp from food animals (Aarestrup et al. 2003;The Merck Manual 1998, eight edition). Table 4 shows Salmonella resistance pattern of ofloxacin at 16% is a reflection that it is least resistant to the organism, whereas the Fluoroquinolones generally have been previously known to have a good sensitivity to Salmonella (The Merck Manual 1998, eight edition). 100% resistance of Salmonella from poultry sources to augmentin is in line with previous works (Ezekiel et al. 2011) even though augmentin has been known to be of variable resistance. Tetracycline have repeatedly shown high levels of resistance of 90%-100% (Sakaridis et al. 2011) to which the work carried out was in agreement.

E. coli antibiotic sensitivity

Resistance patterns to tetracycline and gentamicin were high compared to previous works which showed lower resistance (Musgrove et al. 2006). Resistance of 59% Nalidixic acid and 28.3% gentamicin obtained from research is almost similar to 70.3% Nalidixic acid and 24.3% gentamicin observed in isolates of poultry meat in Saudi Arabia (Altalhi et al. 2010). Augmentin has variable resistance to the E. coli but work showed 100% resistance and Ofloxacin which is known to have a good sensitivity to E. coli, showed the lowest resistance in work. From documented research, E. coli resistance to ciprofloxacin has been found to be higher in broilers than the microorganisms isolated from other sources such as pigs. This might be due to the widespread use of these antimicrobial agents (Miranda et al. 2008). The fluoroquinolones are active against a wide range of bacteria including enteric bacteria but are gradually lossing this ability (Aarestrup et al. 2003) hence of increasing    public health concern. Of necessity is the Veterinary therapeutic use of antimcrobials. However, because treated chickens are sent to the slaughter house immediately after the withdrawal period of antimicrobial agents used, resistant bacteria could be a risk to public health after the slaughter and processing of such birds. It therefore seems necessary to extend the withdrawal period after antimicrobial therapy (Miranda et al. 2008).

Conclusion

1. This study has revealed that imported poultry meat in Ibadan markets are contaminated with Salmonella and E. coli at levels higher than those obtained locally from a processing plant. 2. Salmonella and E. coli are gradually gaining more resistant to antibiotics.

Recommendation

1. Standards of food quality as regards imports of meat and meat-products should be setup and demand for quality certification of products shown before the products are allowed into the country. These standards should have a legal backing and should stand for all imports of raw and ready-to-eat foods. 2. It is important that the use of the HACCP (Hazard Analysis Critical Control point) approach, based on the use of multi-functional strategies (combining the innovative use of sanitizers and modern disinfection techniques) and supervised by professional food handlers and food regulators with a visionary commitment by management from the production, through the processing, preservation, handling and final preparatory stages, be imposed to help eliminate or reduce significantly the prevalence of Salmonella, Escherichia coli and other food-borne pathogens/contaminants and the consequent food poisoning in the society. 3. The indiscriminate use of antibiotics should be cautioned because antibiotics will soon completely loose their effectiveness against microorganisms especially as Escherichia coli acquires antimicrobial resistance faster than other conventional bacteria due to increase resistance. 4. Washing of hands properly before sales of meat or usage of proper clothing such as hand gloves, nose masks and head covers. 5. Proper cold-preservation of meat will prevent multiplication of contaminants. 6. Cooking at high temperatures of 100°C will help to eliminate pathogens before consumption.

Figure 1
1Aerobic plate counts and Enterobacteriaceae counts of chicken meat in Ibadan metropolis area.

Figure 2
2Aerobic plate counts and Enterobacteriaceae counts of turkey meat in Ibadan metropolis area.

Figure 3
3Distribution between Aerobic Plate Counts (APC) andEnterobacteriaceae counts (ENT) in poultry from retail market.

Figure 4
4Aerobic plate counts and enterobacteriaceae counts in chicken from retail markets and processing plant.

Figure 5
5Salmonella spp. and Escherichia coli in chicken from Processing plant and Retail market.

b. Sub-cultivation: Presumptive pinkish colonies (coliform) obtained from incubated plates were subcultivated unto the fresh MCA plates. Plates were labelled, inverted and incubated overnight (18-24 hours) at 37°C. c. Plating on Nutrient agar: Isolates were transferred into nutrient slants, labelled and incubated overnight (18-24 hours) at 37°C. On removal from the incubator, slants were stored in a refrigerator at 4°C, ready for biochemical analysis.Biochemical tests

Catalase test, sugar fermentation using TSI (LabM, Uk),
Mannitol test and Gram staining were carried out for
Salmonella spp. while catalase test, sugar fermentation
using TSI (LabM, Uk), Kovac's test and Gram staining
were employed for Escherichia coli isolates.

Table 1
1Aerobic plate counts and enterobacteriaceae counts for chicken meat in Ibadan metropolisIbadan LGA
Sample
size

Aerobic plate
count cfu/g

Enterobacteriaceae
count cfu/g

n
Mean ± SD
Mean ± SD

Ibadan North
16
7.45 ± 0.48
6.63 ± 2.42

Ibadan North-West
5
7.80 ± 0.37
6.55 ± 1.76

Ibadan North-East
8
7.57 ± 0.42
5.97 ± 2.78

Ibadan South-West
14
7.88 ± 0.61
7.33 ± 1.08

Ibadan South-East
10
6.81 ± 1.79
6.90 ± 1.26

Total
53
7.49 ± 0.94
6.76 ± 1.93

key: LGA = Local Government Area, n = sample numbers collected,
SD = standard deviation, cfu = colony forming unit, g = gram.

Table 2
2Aerobic plate counts and enterobacteriaceae counts for turkey meat in Ibadan metropolisIbadan LGA
Sample
size

Aerobic plate
count cfu/g

Enterobacteriaceae
count cfu/g

n
Mean ± SD
Mean ± SD

Ibadan North
18
7.16 ± 1.40
6.87 ± 2.00

Ibadan North-West
5
7.55 ± 0.49
7.18 ± 0.38

Ibadan North-East
5
7.53 ± 0.54
5.88 ± 2.30

Ibadan South-West
11
7.68 ± 0.46
6.63 ± 2.57

Ibadan South-East
7
7.51 ± 0.86
6.90 ± 1.77

Total
46
7.42 ± 1.00
6.74 ± 2.00

key :

Table 3
3Aerobic plate counts and enterobacteriaceae counts in chicken meat from processing plant in comparison to those from retail marketIbadan metropolis Sample
size

Aerobic plate
count cfu/g

Enterobacteriaceae
count cfu/g

n
Mean ± SD
Mean ± SD

Processing plant
53
4.18 ± 1.47
3.99 ± 2.53

Retail shops
53
7.50 ± 0.94
6.76 ± 1.93

Total
106
5.84 ± 2.07
5.38 ± 2.64

n = sample numbers collected, SD = standard deviation, cfu = colony forming
unit, g = gram.

Table 4
4Antimicrobial susceptibility pattern for Salmonella enterica spp. (n = 45) Key: No. -numbers of isolates, Aug (Augmentin), Ofl (Ofloxacin),Gen (Gentamicin), Nal (Nalidixic acid), Nit (Nitrofurantoin), Cot (Cotrimaxazole) and Tet (Tetracycline).Antibiotics
(μg)

Totally
sensitive

Intermediate
Resistant

No.
No.
No.

Aug (30)
0
0%
0
0%
45
100%

Ofl (5)
33
73%
5
11%
7
16%

Gen (10)
23
51%
9
20%
13
29%

Nal (30)
0
0%
12
27%
33
73%

Nit (200)
10
22%
10
22%
25
56%

Cot (25)
3
7%
0
0%
42
93%

Tet (25)
0
0%
3
5%
42
93%

Table 5
5Antimicrobial susceptibility pattern for Escherichia coli (n = 46) Key: n -numbers of isolates, Aug (Augmentin), Ofl (Ofloxacin), Gen (Gentamicin), Nal (Nalidixic acid), Nit (Nitrofurantoin), Cot (Cotrimaxazole) and Tet (Tetracycline).Antibiotics
(μg)

Sensitive
Intermediate
Resistance

No.
No.
No.

Aug (30)
0
0%
0
0%
46
100%

Ofl (5)
29
63%
6
13%
11
24%

Gen (10)
14
30.4%
19
41.3%
13
28.3%

Nal (30)
5
11%
14
30%
27
59%

Nit (200)
17
37%
5
11%
24
52%

Cot (25)
3
7%
2
4%
41
89%

Tet (25)
8
17%
0
0%
38
83%

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Competing interestsThe authors declare that they have no competing interests.Authors' contributionsBoth authors read and approved the final manuscript.
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Adeyanju and Ishola: Salmonella and Escherichia coli contamination of poultry meat from a processing plant and retail markets in Ibadan. Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the fi eld 7. Oyo State, Nigeria3139SpringerPlus. Retaining the copyright to your article Submit your next manuscript at 7 springeropen.comCite this article as: Adeyanju and Ishola: Salmonella and Escherichia coli contamination of poultry meat from a processing plant and retail markets in Ibadan, Oyo State, Nigeria. SpringerPlus 2014 3:139. Submit your manuscript to a journal and benefi t from: 7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the fi eld 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com