Investigating the Inhibitory Effect of Lactic Acid on Biofilm Production by Raw Chicken Meat Campylobacter spp. Isolates in Pure and Mixed Cultures ^†

Abstract

Campylobacter spp. are the main cause of foodborne gastroenteritis worldwide, and the biofilm growth mode seems to play a key role in their prevalence. In this work, the effect of lactic acid (LA) on planktonic growth and biofilm production by eight Campylobacter spp. raw chicken meat isolates was investigated using polystyrene and stainless steel as the abiotic substrata. Results revealed that the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum biofilm inhibitory concentration (MBIC) values of LA against the Campylobacter isolates ranged from 1024 to 4096 μg/mL depending on the isolate, mode of growth (planktonic vs. biofilm; single vs. mixed culture), and the growth medium. Overall, the results of this work offer insights into the biofilm control of a pathogen of public health importance.

1. Introduction

Campylobacter spp. are zoonotic agents in broiler chickens and their products, as well as unpasteurized milk, which is their main reservoir [1,2]. Chickens (Gallus gallus domesticus) hence act as their most common hosts, and in combination with the incorrect handling of these products (usually insufficient cooking, as well as cross-contamination events), the pathogenic bacteria can end up on the consumer’s plate [3]. Campylobacteria are Gram-negative microaerophilic bacteria with a spiral (curved) or rod-like shape [4]. According to the European Food Safety Authority (EFSA), Campylobacter jejuni and Campylobacter coli are the most frequently reported species in cases of campylobacteriosis [2].

Campylobacter spp. are fragile and fastidious in their growth requirements but, paradoxically, they can be easily transmitted from animals to humans through the food production chain [5]. The mode of biofilm growth is suggested as a key survival and persistence mechanism used by them [6]. Interestingly, Campylobacter spp. cannot grow and multiply in food during processing and storage, as happens with most other foodborne pathogenic bacteria, since the prevailing conditions (e.g., aerobiosis and temperature) are usually unfavorable for them [7]. An important role in their survival and eventual dominance against other pathogens should thus be played by their ability to attach to food-related surfaces and their inclusion in multi-species biofilms [8,9,10]. Macromolecules such as food constituents can influence the attachment of bacteria to surfaces, whereas it has been reported that the presence of proteins such as albumin, gelatin and casein can influence the initial attachment of bacteria to some surfaces [11,12,13]. However, it seems that there are no sufficient relevant data regarding Campylobacter spp. in single or mixed cultures. Additionally, chicken juice has been suggested to promote the attachment of C. jejuni by creating a conditioning film on the abiotic surface, while at the same time it seems like a suitable laboratory model with which to study Campylobacter biofilm formation as it mimics the conditions present in the environment of slaughterhouses [14,15].

Although it is difficult, if not impossible, to completely get rid of campylobacteria, there are still many physical and chemical strategies that can be used to limit their prevalence. These can be employed at different stages of the food production chain [16]. During food animal processing, for instance, organic acids may be used to remove pathogens from carcasses and decrease their microbial load. Organic acids are also used as acidifiers in poultry drinking water and as antimicrobial feed additives, having at the same time a positive effect on the good functioning of the poultry digestive system. Noteworthily, poultry carcass treatment with 2% w/v (20 mg/mL) lactic acid (LA) is estimated to reduce the risk of Campylobacter infection in humans by between 37 and 56% [17].

In this work, the inhibitory effect of LA on planktonic growth and biofilm production by eight Campylobacter spp. raw chicken meat isolates (five C. jejuni and three C. coli) on model polystyrene (PS) surfaces was investigated. The inhibitory effect of this acid on a mixed culture biofilm (composed of three different isolates) was also determined using six-well PS microplates and stainless steel (SS) coupons as the abiotic substrata.

2. Methods

2.1. Preparation of Sterile Chicken Juice (CJ)

Minced raw chicken (≈300 g) was purchased from a local supermarket and immediately transported to the laboratory. In a stomacher bag, 250 g of minced meat were weighed and 250 mL of sterile deionized water were then added (in a 1:1 dilution). The mixture was homogenized in a stomacher (BagMixer^® 400; Interscience, Saint Nom la Bretêche, France) for 3 min, then aliquoted into 50 mL plastic Falcon tubes and centrifuged at 7000× g for 12 min at 4 °C (to remove animal tissue sediment). Following this, the supernatants were carefully removed from each tube and collected together into a glass beaker. The aqueous mixture was initially filtered through paper filters (200 g/m²; diameter 55 mm; Munktell Filter AB, Falun, Sweden) using a Buchner funnel to remove the largest aggregates. The filtrate was then aseptically filtered through microbiological filters (pore diameter 0.22 µm; SFCA-22E-050, Labbox Labware S.L., Barcelona, Spain) and stored at −80 °C.

2.2. Campylobacter Isolates and Preparation of Their Working Cultures

The raw chicken meat Campylobacter isolates used in this work belonged to the species C. jejuni (n = 5) and C. coli (n = 3). Some other critical information on these isolates is provided in

Table 1. Campylobacter raw chicken meat isolates used in this study and their relevant info.

Isolate Code	Species	Other Information	Poultry Isolation Origin
CAMP 1_005	C. coli	strong BP 2, MDR 3	wings
CAMP_022	C. jejuni	strong BP, MDR	minced meat
CAMP_025	C. coli	strong BP, MDR	neck
CAMP_048	C. jejuni	strong BP	souvlaki
CAMP_083	C. coli	weak BP, high resistance to ERY 4	thigh
CAMP_091	C. jejuni	weak BP, high resistance to ERY	wings
CAMP_114	C. jejuni	moderate BP, MDR	neck
CAMP_130	C. jejuni	weak BP, MDR	wings

¹ Campylobacter; ² biofilm producer; ³ multidrug resistance; ⁴ erythromycin.

. Before their experimental use, the isolates were stored long-term at −80 °C in Mueller–Hinton (MH) broth (CM0405, OXOID Limited, Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 5% v/v laked horse blood (HB) (SR0048C, Thermo Fisher Scientific Inc.) and 20% v/v glycerol (Merck KGaA, Darmstadt, Germany). When needed for the assays, each isolate was streaked on the surface of MH agar (AGMH-00P-500, Labbox Labware, S.L., Barcelona, Spain) and incubated at 42 °C for 24 h under microaerophilic conditions (6.2–13.2% O₂, 2.5–9.5% CO₂; Oxoid CampyGen 2.5L Sachet; CN0025A, Thermo Fisher Scientific Inc.) (primary precultures). Secondary precultures were prepared by inoculating a biomass of 5 to 10 colonies from each primary preculture into 2 mL of fresh MH–HB broth and then incubating them at 42 °C for 24 h under microaerophilic conditions. Working cultures were prepared by transferring 200 μL of each secondary preculture to 1800 μL of fresh MH–HB and then incubating them at 42 °C for 24 h under microaerophilic conditions (thereby achieving a final concentration of ca. 10⁸ CFU/mL).

2.3. Determination of Minimum Inhibitory and Bactericidal Concentrations (MICs and MBCs) of LA against Planktonic Campylobacter Bacteria

The MIC of LA against the planktonic cells of each Campylobacter isolate was determined using the broth microdilution method as previously described, with slight modifications [18]. Briefly, bacteria from the final working cultures (ca. 5 × 10⁵ CFU/mL) were incubated in two different nutrient broths, i.e., either MH or MH–HB broth, at 42 °C for 48 h under microaerophilic conditions. For each broth, seven different concentrations of LA were tested ranging from 4096 to 64 μg/mL (two-fold dilutions). The MIC of LA was considered its lowest concentration, resulting in no visible bacterial growth. The absence of growth was confirmed through the lack of increase in the absorbance of the medium in the case of MH broth, whereas in the case of MH–HB broth, this was verified by the naked eye through observing a change in color of the medium from red to brown. Resazurin sodium salt (B21187; Alfa Aesar; Haverhill, MA, USA) was also used at a concentration of 0.01% w/v, as an indicator of metabolic activity in the case of MH broth [19]. To calculate the MBC, 10 μL of broth cultures were aspirated from all the non-growth wells of the MIC assay and spotted (in duplicate) on MH agar plates, which were then incubated at 42 °C for 48 h under microaerophilic conditions. For each bacterial isolate, the MBC of the LA was determined as its lowest concentration that reduced the initial inoculum (ca. 5 × 10⁵ CFU/mL) by more than 99.9% (no appearance of colonies). These experiments were repeated three times starting with independent bacterial cultures.

2.4. Determination of Minimum Biofilm Inhibitory Concentrations (MBICs) of LA against Single and Mixed Campylobacter Cultures

2.4.1. Determination of MBICs of LA against Single Cultures

The MBIC of LA against the biofilm growth of each Campylobacter isolate was determined using the crystal violet (CV) staining assay as previously described [20]. For this, bacteria were initially left to form biofilms on 96-well PS microtiter plates (transparent, flat, Cat. No. 30096, SPL Life Sciences, Gyeonggi-do, Republic of Korea) for 48 h in MH broth supplemented with 5% (v/v) chicken juice (MH–CJ) at 42 °C under microaerophilic conditions and in the presence of seven different LA concentrations (two-fold dilutions ranging from 4096 to 64 μg/mL). At the end of incubation, for each bacterial isolate and LA concentration, the accumulated biomass in each well was quantified following its staining with CV (0.1% (w/v)), the solubilization of the bound dye with an ethanol-acetone mixture (80:20, v/v), and absorbance measurements of the resulting solution at 590 nm (A_590nm) using a multimode microplate reader (Tecan Spark^®, Tecan Group Ltd., Männedorf, Switzerland). As a positive control for biofilm growth, wells containing inoculated ΜH–CJ without LA were used, whereas wells containing uninoculated MH–CJ were used for the negative control. For each bacterial isolate, the MBIC of the LA was determined as its lowest concentration that completely inhibited biofilm formation (the biomass accumulated was not significantly different from that of the negative control). These experiments were repeated three times starting with independent bacterial cultures.

2.4.2. Determination of MBICs of LA against Mixed Cultures

The procedure described in Section 2.4.1. was also followed to determine the MBICs of LA against three different mixed Campylobacter cultures (consortia), each composed of three isolates (

Table 2. The three different Campylobacter consortia, each composed of three isolates. The six different isolates of these consortia were divided into three different groups (A–C) depending on their drug resistance and biofilm-forming phenotypes.

Consortium Code	Group A 1	Group B 2	Group C 3
CONS1	CAMP_130	CAMP_083	CAMP_048
CONS2	CAMP_130	CAMP_091	CAMP_022
CONS3	CAMP_130	CAMP_083	CAMP_005

¹ MDR; ² no MDR, with high-level resistance to ERY; ³ strong biofilm producing capacity.

). In this case, nine different LA concentrations (two-fold dilutions ranging from 16,384 to 64 μg/mL) were tested. The selected Campylobacter isolates (n = 6) were divided into three different groups based on MDR character (Group A), high ERY resistance but not MDR (Group B), and strong biofilm production capacity (Group C). In addition, isolates with different macroscopic colony characteristics were selected per group.

2.5. The Inhibitory Effect of LA on Biofilm Production by a Mixed Campylobacter Culture on PS and SS Surfaces

The inhibitory effect of LA against a selected mixed Campylobacter culture (i.e., CONS1) was further tested following the procedure described thereafter. In this treatment, six-well PS microplates and SS coupons (30 × 10 × 1 mm, type AISI 304; placed vertically into glass tubes) were used as the abiotic substrata. For both types of substrata, 5 mL of growth medium (MH–CJ) were used to fill either each PS well or each glass tube. Four different LA concentrations were examined (two-fold dilutions ranging from 4096 to 512 μg/mL). After 48 h of static incubation at 42 °C under microaerophilic conditions, planktonic and biofilm cells were quantified via serial decimal dilutions in quarter-strength Ringer’s solution (Lab M, Heywood, Lancashire, UK) and subsequent inoculation through spreading of MH-HB agar plates. More specifically, to detach and enumerate the biofilm cells, following the removal of planktonic suspension, each well was washed twice with quarter-strength Ringer’s solution, and after being filled with 5 mL of the same solution, it was thoroughly scratched with a plastic sterile pipette tip to remove the biofilm cells. Concerning the SS coupons, each of them was also washed twice with quarter-strength Ringer’s solution, then placed into a Falcon tube containing 5 mL of the same solution and 10 glass beads (3 mm diameter), and vortexed for 2 min.

3. Results and Discussion

The inhibitory and lethal effects of LA against planktonic populations of eight wild-type Campylobacter isolates, grown in MH broth supplemented or not with 5% laked horse blood, under microaerobic conditions for 48 h at 42 °C, were initially tested in this study. The reason for selecting two different broths was because Campylobacter spp. usually grow better in nutrient media supplemented with blood; however, there is no standardized protocol for the use of blood in the broth microdilution method for MIC determination. It should also be noted that in the case of LA treatments in the presence of blood, it was not possible to obtain reliable results from the spectrophotometric data. However, in this case, we were still able to accurately determine the endpoint MICs at those LA concentrations where a change in broth color from red to brown was observed (always comparing to the negative control).

Table 3. MIC, MBC and MBIC values of LA against the eight Campylobacter isolates and the three different consortia.

Campylobacter/Consortium Code		MIC 2	MBC 3	MIC	MBC	MBIC 4
	Species/Isolates	μg/mL
		in MH 5		in MH -HB 6		in MH -CJ 7
CAMP 1_005	C. coli	1024	1024	2048	2048	1024
CAMP_022	C. jejuni	1024	1024	2048	2048	1024
CAMP_025	C. coli	1024	1024	1024	1024	1024
CAMP_048	C. jejuni	2048	2048	2048	2048	1024
CAMP_083	C. coli	1024	2048	2048	2048	2048
CAMP_091	C. jejuni	2048	2048	2048	2048	1024
CAMP_114	C. jejuni	1024	1024	2048	2048	1024
CAMP_130	C. jejuni	1024	1024	2048	2048	2048
CONS1	CAMP_048/083/130					4096
CONS2	CAMP_022/091/130					4096
CONS3	CAMP_005/083/130					4096

¹ Campylobacter; ² minimum inhibitory concentration; ³ minimum bactericidal concentration; ⁴ minimum biofilm inhibitory concentration; ⁵ Mueller–Hinton broth; ⁶ MH with 5% v/v laked horse blood; ⁷ MH broth with 5% v/v chicken juice.

presents the results of MIC/MBC determination for each one of the eight Campylobacter isolates. These results reveal that in general, the presence of blood seems to favor the resistance of campylobacteria to LA treatment, with MIC values recorded equal to 1024 μg/mL for six of the eight tested isolates incubated in blood-free MH broth; on the other hand MIC values equal to 2048 μg/mL were recorded for seven of the eight tested isolates incubated in MH broth with blood. However, these observed differences in MIC values are probably without any important practical effect, considering that LA is commonly applied at much higher concentrations (>5000 μg/mL; 0.5% v/v) in antimicrobial treatments encountered in poultry processing [17,21]. For almost all the Campylobacter isolates, the MICs of LA were equal to the MBCs, indicating its strong bactericidal action.

Regarding the inhibitory effect of LA on biofilm production by the Campylobacter isolates grown in monoculture, the recorded MBIC values were 1024 μg/mL for six of the eight isolates, while for the other two of them (CAMP_083 and CAMP_130), the required LA concentration to inhibit their biofilm growth was double and equal to 2048 μg/mL (Table 3). It is worth noting that these results do not reveal any relationship between biofilm forming capacity (weak, moderate, or strong) of a given isolate and the LA biofilm-inhibitory action against it. In the case of mixed Campylobacter biofilm cultures (CONS1, CONS2, and CONS3), a LA concentration of 4.096 μg/mL was always required to inhibit the growth of biofilms for all three consortia (Table 3). Alarmingly, this denotes the favoring effect of inter-strain interactions on the resistance of mixed-culture biofilms to LA.

Figure 1 presents the biofilm populations (Log₁₀CFU/mL) for each one of the three isolates (CAMP_130, CAMP_083 and CAMP_048) of the mixed Campylobacter culture CONS1 incubated in MH-CJ in the presence of four LA concentrations, on either PS (six-well microplates) or SS (coupons) surfaces, under microaerophilic conditions for 48 h at 42 °C. For both surfaces, CAMP_130 and CAMP_083 isolates appeared to dominate over the CAMP_048 isolate at the two highest LA concentrations that were applied (2048 and 4096 µg/mL). This is an interesting observation and is probably due to the fact that the MBIC value of LA against CAMP_048 was lower than that observed against the two other isolates, thus indicating its higher sensitivity to LA. In addition, the competition that may develop between the different isolates under the mixed culture conditions, mainly for available nutrients, may also account for this observation, as has been previously reported for some other bacterial species [22].

4. Conclusions

Overall, the results of this work offer insights into the biofilm control of a pathogen of public health importance.