LOW NUTRIENT R2A CULTURE MEDIUM FOR BACTERIAL ENUMERATION FROM POULTRY FEEDS

LOW NUTRIENT R2A CULTURE MEDIUM FOR BACTERIAL ENUMERATION FROM POULTRY FEEDS

K.G. MACIOROWSK11.3, S.D. PILLAI', F.T. JONES' and S.C. RICKEi1v4

'Poultry Science Department Texas A&M University

College Station, TX 77843-2472

2Center of Excellence for Poultry Science University of Arkansas Fayetteville, AR 72701

Accepted for Publication March 14, 2002

ABSTRACT

Poultry feed matrices can be particularly stressful to bacterial populations and limit recovery and accurate enumeration of viable organisms. The objectives in this study were to enumerate bacterial populations with either tryptic soy plate medium or an R2A minimal nutrient medium from commercial poultry and turkey dry feeds and compare enumerations from the two media with feed composition. Total population estimates from tiyptic soy plates vaned between 3.7 and log,, 5.4 CFU per g feed and depended upon poultry feed source (P € 0.05). R2A plate counts varied between 2 .7 and log,, 5.4 CFU per g feed, but were not significantly different among poultry feed sources. Feed R2A plate count populations were significantly correlated (P € 0.01) with tryptic soy plate count populations enumerated from the feed sources, but not protein and fat content of the feed source. It is apparent that bacterial counts recovered by minimal R2A medium are similar to enumerations using tryptic soy plate medium and that R2A medium could be substituted for tryptic soy plate medium for bacterial enumeration in poultry feeds.

INTRODUCTION

Feed can potentially serve as a major vehicle for introducing microorgan- isms to poultry and has been identified as a source for fungal mycotoxins and

Current address: Dr. Kenneth G. Maciorowski, Department of Agriculture and Natural Resources, 1200 North DuPont Highway, Delaware State University, Dover, DE 19901 Address for correspondence: Dr. Steven C. Ricke, Poultry Science Department, 2472 TAMU, College Station, TX 77843-2472. TEL: (979) 862-1528; FAX: (979) 845-1921; E-mail: sricke@poultry .tamu.edu

Journal of Rapid Methods and Automation in Microbiology 10 (2002) 59-68. All Rights Reserved. "Copyrighr 2002 by Food & Nutrition Press, Inc., Trumbull, Connecticut. 59

60 K.G. MACIOROWSKI, S.D. PILLAI, F.T. JONES and S.C. RICKE

foodborne Salmonella spp. (Williams 1981; Tabib et al. 1981; Leeson and Marcotte 1993). Consequently, assessment of feed microbiological quality has become an ongoing concern (Leeson and Marcotte 1993). However, effective and representative recovery of the diverse microflora present in poultry feeds is dependent on several factors. The predominant microbial species in animal feed can vary between different feed types and processing conditions. Loken et al. (1968) reported that Micrococcus and Bacillus predominate in protein feed supplements, while Cox et al. (1983) isolated Enterobacteriacae such as Enterobacter agglomerans, Ent. cloacae, and Klebsiella pneumoniae from commercial poultry diets. In addition, poultry feed matrices can be particularly stressful to bacterial populations. For example, dry poultry feeds are typically marketed at low water activities ranging from 0.5 to 0.7 and this along with other factors such as temperature can prevent multiplication of salmonellae and influence survival (Liu et al. 1969; Carlson and Snoeyenbos 1970; Juven et al. 1984). Feed treatments used to decrease microbial contamination such as pelleting (Veldman et al. 1995), irradiation (Leeson and Marcotte 1993; Ha et al. 1997a) or acid addition (Hinton and Linton 1988; Ha et al. 1997b; 1998a) may also adversely affect bacterial populations in feed and therefore interfere with enumeration on selective media (Kafel 1981).

Accurate enumeration of bacteria in environmental matrices such as poultry feed depends on the utility of the media to effectively recover viable bacteria that may be in various physiological states of growth and survival. These bacteria may be difficult to culture by standard methods and require culture media that supports recovery of bacteria that exist under the more stringent conditions associated with feeds. A low nutrient agar plate agar medium (R2A agar) for recovery of bacteria was developed by Reasoner and Geldreich (1985) to assess microbial quality of water sources by public health and water treatment laboratories (Horgan el al. 1999). Low nutrient R2A agar medium has been utilized for bacterial enumeration from clinical settings, biofilters, drinking water, aerosols, and sewage sludges (Pernitsky et al. 1995; Heidelberg et al. 1997; Jayasekara et al. 1998; Horgan et al. 1999; Moll et al. 1999; van der Linde et al. 1999). In addition, higher numbers of culturable Salmonella typhimurium were recovered on R2A medium versus other media after bacterial cells had been subjected to 9 weeks of desiccation and starvation in a model system (Lesne et al. 2000). This indicates that R2A medium may also be useful for recovery of bacteria from dry matrices such as poultry feed mixes and ingredients (Carlson and Snoeyenbos 1970; Juven et al. 1984). Our objectives in this study were to enumerate bacterial populations with either tryptic soy plate medium or R2A medium from commercial poultry and turkey dry feeds and compare enumerations from the two media with feed composition.

R2A MEDIUM FOR BACTERIAL ENUMERATION FROM POULTRY FEEDS 61

MATERIALS AND METHODS

Poultry Feeds and Composition Analysis

Twelve poultry diets were collected by the Department of Poultry Science at the University of Arkansas and coded ‘A’ through ‘L’. Diets were obtained from commercial sources and were typical starter chicken or turkey corn- soybean based diets supplemented with small amounts of protein byproducts. Protein, fat and ash were determined according to AOAC standard methods (AOAC 1995). Protein was determined on 50 mg duplicate samples with a Fisons model NA-2000 (Fison Instruments, Beverly, MA) and AOAC method 968.06. Fat content was determined using 2 g duplicate samples extracted with 50 mL of ethyl for hours as described in AOAC method 942.05. Ash contents were determined on single samples.

Bacterial Enumerations

The bacterial populations from all poultry diets in this study were enumerated as follows. Ten grams of each dietary sample were added to 100 mL of tryptic soy broth in a sterile Stomacher bag and manually shaken for 2 min to represent the ‘0’ dilution. Ten-fold serial dilutions were made in 0.2% peptone and duplicate 0.5 mL aliquots were inoculated onto the respective plate media and spread with approximately 10 sterile glass beads contained in each plate (100 x 15 mm plates). For enumeration of the bacterial populations in poultry diets, either R2A medium (Difco Laboratories, Sparks, MD) or tryptic soy agar medium (TSA; Difco) was used. The plates were incubated at 25C for 3 days and colony forming units (CFU) were enumerated.

Statistical Analysis

Feed composition analysis for protein, fat and ash were expressed as percentages (w/w). Protein and fat were statistically analyzed by least square mean separations using the PDIFF option and the generalized linear models (GLM) procedure of SAS (Statistical analysis version 6.04, 988; SAS Institute, Cary, NC). Protein and fat percentages were considered significant at P < 0.05. Plate enumerations were expressed as colony forming units (CFU) per g of feed for analysis and converted using a log,,, function for analysis. Model adequacy was determined by linear regression using the GLM procedure of SAS and considered significant at the P < 0.05 level. Differences in least squares means for plate enumerations were determined using a Fisher’s protected least significant difference test (Steel and Torrie 1980) and the PDIFF option of SAS. Mean differences were declared significant at the P < 0.05 level. Average log,, plate enumerations from TSA and R2A bacterial isolations were analyzed across


all feed sources and feed protein and fat composition for Pearson correlation by the correlation (CORR) procedure of SAS. Correlations were declared significant at the P < 0.01 level.

RESULTS AND DISCUSSION

Composition of poultry feeds are presented in Fig. 1. Protein levels for the 12 starter chicken or turkey corn-soybean based commercial diets represented a variety of protein levels ranging from 29.6% of the total weight of the diet (w/w) to 15.9%. Feed samples from the source designated “I” contained the greatest level of protein (P < 0.05) followed by feed source “F” and “J” while sources “L” and “E” contained the least amount of protein (P < 0.05) with the remainder of the diets containing mid-range levels of protein. Fat content ranged from 10.3 to 3.7% w/w but the pattern among the different diets was somewhat different than protein content. Feed samples from the source designated “D” contained the greatest level of fat ( P < 0.05) followed by feed source “A” while sources “K”, “J” and “E” contained the least amount of fat (P < 0.05) with the remainder of the diets containing mid-range levels of fat. Ash contents were not statistically analyzed but ash ranged from 9.2 to 3.7% w/w with diet “D” containing nearly twice as much ash as most of the other diets.

Bacterial population enumerations of the poultry diets using TSA plate medium are shown in Fig. 2. When bacteria in poultry diets were enumerated, total number of isolates depended upon dietary diet source (P < 0.05). Total bacterial populations ranged between log,, 3.7 CFU per gram feed and log 5.4 CFU per gram feed. Diet source “C” yielded the lowest bacterial populations enumerated on TSA plates ( P < 0.05), with log 3.7 CFU per gram feed, while all other diets were significantly higher, ranging between lo3 to lo5 CFU per gram feed. The bacterial populations enumerated in the current study are less than the minimum of 1 x lo7 CFU per gram detected on nutrient agar by Banerjee and Shetty (1992) for mixed feeds obtained from poultry farms and poultry feed mills located on the Andaman and Nicobar islands in the Bay of Bengal. However, bacterial enumerations on typical U.S. poultry diets have generally yielded lower numbers of bacterial contaminants. Chen et al. (1979) observed a range of lo4 to lo6 CFU per gram for poultry diets. Ha et al. (1995, 1997a, b; 1998a, b) using either plate count or tryptic soy agar media detected bacterial populations ranging from lo4 to lo7 CFU per gram in a soybean meal- based poultry mash and poultry feeds containing soybean meal, meat and bone meal or fishmeal as protein sources. Although it is difficult to compare across individual studies, it has been shown previously that bacterial numbers can vary greatly with their ingredient sources (Chen et al. 1979) and higher levels of moisture can lead to growth of some organisms such as salmonellae (Carlson and Snoeyenbos 1970).

R2A MEDIUM FOR BACTERML ENUMERATION FROM POULTRY FEEDS 63

30

h 25 P 3. 20 + C a 15

at E Il 10

5

0 A B C D E F G H I J K L

Poultry Diet

Improtein Dfat mash1

FIG. 1. COMPOSITION OF POULTRY FEED SOURCES Protein and fat percentages represent an average of two determinations. Different small letsers

above respective bars in graph represent significant differences (P < 0.05) in protein percentages. Different capital letters above respective bars in graph represent

significant differences (P < 0.05) in fat percenrages.

'1

A B C D E F G H I J K L

Poultry Diet FIG. 2. TOTAL TSA PLATE ENUMERATIONS FOR POULTRY FEED SOURCES

Values represent an average of two determinations. Different capital letters above bars in graph represent significant differences (P < 0.05) in log,, CFU.

64 K.G. MACIOROWSKI, S.D. PILLAI, F.T. JONES and S.C . RICKE

Bacterial populations enumerated on R2A plates are shown in Fig. 3. Bacterial enumerations were not significantly influenced by poultry feed source (P > 0.05). R2A plate counts ranged from 10’ to lo5 CFU per g feed with poultry feed source “H” yielding nearly twice the log,, compared to feed source “C”. In general, similar trends were observed among feed sources for bacterial populations enumerated on R2A medium as was observed with TSA plate counts. Feed sources “H” and “D”, tended to yield higher counts than the other feed sources while feed source R2A bacterial populations from poultry feed sources “F”, “J”, “ I” , and “C” tended to be lower. As a general trend, most of the bacterial isolations from R2A plates yielded slightly lower population enumerations that TSA plate counts, with the exception of feed sources “A” and “L” which were slightly higher than TSA plate counts. Bacterial population estimates vary considerably in reports on isolations from feed and depending on the type of bacterial population enumerated. Tabib et al. (1981) detected between 5 to nearly lo6 coliforms per g of broiler and turkey feed. Cox et al. ( 1983) determined the average populations of Enterobacteriacae in commercial poultry feed to be 4.1, 0.8, and 1.8 log CFU per gram of mash, pelleted, and meal samples, respectively. Veldman et al. (1995) noted that Enterobacteriacae populations in pelleted feed may be as great as lo5 CFU per gram. ZabaIa Diaz et al. (1999) estimated plate agar counts approximately lo4 CFU per gram for either fresh poultry feeds or poultry feeds stored at 4C for several months. However, in this same study when they used a highly selective medium, indigenous E. coli populations were usually no more than 10’ CFU.

It appears that bacterial quality is highly variable given the wide range of bacterial counts observed in this study among feeds. There are some indications from previous studies that feed composition can influence bacterial counts and survivability. Chen et al. (1979) observed that not only did bacterial counts vary greatly with ingredient sources but fungistatic compounds had no consistent impact on aerobic, anaerobic, mold or yeast populations. Mossel and Koopman (1965) suggested that salmonellae imbedded in proteins and protected by lipids in naturally contaminated dry feeds may be more protected from normally lethal heat treatments than bacterial inocula artificially introduced into feed. More specifically, Dixon and Hamilton (1981) suggested that bacterial pathogen survival in feeds may be influenced by the buffering capacity of the protein source. In an attempt to explain the variation of TSA and R2A bacterial populations recovered from poultry feeds in the current study, respective populations from poultry feed sources were correlated against each other, fat and protein concentrations (Table 1). None of the bacterial populations assayed from the poultry feeds correlated significantly (P > 0.01) with fat or protein content. However, bacterial populations enumerated on R2A plates were significantly correlated with bacterial populations enumerated on TSA plates ( P < 0.01) enumerated from the poultry feed sources. This is in agreement with the limited


effect observed by Ha et al. (1998b) of protein concentration on survival of a Salmonella typhimurium marker strain inoculated into poultry mash with variable protein levels.

6

5 m 3 u - 4 0, r m 0 3 -

2

1

0

A B C D E F G H I J K L

Poultry Diet

FIG. 3. R2A PLATE ENUMERATIONS FOR POULTRY FEED SOURCES Values represent an average of two determinations.

TABLE 1. PEARSON (R’) CORRELATIONS AMONG R2A AND TSA ENUMERATED

BACTERIAL POPULATIONS FROM POULTRY FEED SOURCES

Factor Correlation

R

RZA:TSA 0.716

R2A: fat content 0.143

WA: protein content - 0.269

TSA: fat content 0.030

TSA: protein content - 0.396

Fat: protein contents - 0.005

P value

<0.01 *

< 0.55

< 0.25

< 0.90

< 0.10

< 0 99

* Pearson correlation coefficients with a P < 0.01 were considered significant.

66 K.G. MACIOROWSKI, S.D. PILLAI, F.T. JONES and S.C. FUCKE

In conclusion it is apparent that bacterial counts recovered by minimal R2A medium correlate with recoveries using TSA plate medium but neither are correlated with fat or protein content indicating that the levels of these ingredients has minimal influence on overall bacterial populations. It also appears that at least for commercial poultry feed samples, R2A medium could be substituted for TSA plate medium for bacterial counts. However, slight differences between the recovered populations may also be a factor because of the generally lower bacterial populations recovered from the same feed sources when R2A plate medium is used. It is possible that these differences would become more accentuated if feed bacterial populations became more stressed. It would be of interest to compare these two plating media for recovery of bacterial populations after feeds were stored for long periods or were subjected to treatments known to be deleterious to bacteria such as acid addition or heat treatment.

ACKNOWLEDGMENTS

This research was supported by the Texas Higher Education Coordinating Board’s Advanced Technology Program (Grant # 999902-165), TEX08239 project, and Hatch grant H8311 administered by the Texas Agricultural Experiment Station. The Pilgrim’s Pride (Pittsburg, TX) endowed graduate fellowship and a Heep Foundation Internship supported K.G.M. The authors would like to thank Xin Li for assistance in constructing the figures and statistical analysis for Fig. 1.

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LOW NUTRIENT R2A CULTURE MEDIUM FOR BACTERIAL ENUMERATION FROM POULTRY FEEDS