Serratia, a case of study. A Bolarin Quality & Research Manager of AIM Ibérica S.A.U

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1 Serratia, a case of study A Bolarin Quality & Research Manager of AIM Ibérica S.A.U Abstract A 3-years retrospective study was made on six AI boar farms of Spain and Portugal. One extended semen sample of each boar collection was routinely analyzed for longevity and bacteriology. Longevity was subjectively measured by an experienced technician using a phase contrast microscope. Semen samples were pre-warmed in a water bath during minutes at 34ºC, and then motility was checked once, at hours after collection. Semen samples with more than 50% motility were considered normal. For those samples that showed reduced motility, a bacteriological culture was performed. Cultures were made in blood agar plates, using sterile swabs, and placed during 48h into a warm cabinet at 34-35ºC. Blood agar plates with bacteria growth were sent for bacteriological identification. Simultaneously, boar ejaculates were randomly and routinely analyzed for bacteriology. Antibiogram was randomly performed. A total of 102 semen samples and 26 ejaculates were analyzed, from which 6,86% and 26,92% respectively showed two different bacteria in the cultures, whereas the rest of them showed growth of only one type of microorganism. A total of 31 different bacteria were found in cultures of both extended semen samples and ejaculates. The most common bacteria isolated were Serratia liquefaciens, Serratia marcescens, Staphylococcus spp. and micrococcus spp, representing 26,1% (n=37), 12% (n=17), 5,6% (n=8) and 5,6% (n=8) respectively of all the 142 isolations. In 4 cases (2,8%), the bacteria could not be identified. The isolated Serratia different species represented the 44,44% (n=64). All bacteria isolated were found to be resistant to the Beta-lactamics amoxicilin and ampicilline and to the cephalosporine cefuroxime. Moreover, above 90% of the colonies were also resistant to other common antibiotics present in commercial extenders, such as the aminoglycosides neomicyn and espectinomycine, and the Beta-lactamic amoxicilin-clavulanic acid. The antibiotics contained in the extender used in all AI boar studs of the study were neomicyn and penicillin. To decrease or eliminate bacteria load in doses, and gain time to identify the contamination sources, enrofloxacine was temporary added to extender immediately after growth detection. In 123 cases out of 128, microbiological contamination was controlled and totally eliminated, even if the bacteria were found to be resistant to enrofloxacine, neomicyn and penicillin. Whenever bacterium was not eliminated, ceftiofur was successfully applied to the extender. Bacteria were introduced in production line from multiple sources, including animals, bad collection procedures, distillated water supply, personnel and several times, unknown sources. These findings encouraged the application of a custom monitoring protocol for each of the six boar studs, to prevent microbiological contamination, and decrease bacteria load in all critical points. Key words: boar, semen, bacteria, serratia, antibiotics.

2 1. Introduction Artificial insemination (AI) has become a common practice in swine production in the last two decades. The market trend is production semen doses in big professional AI boar studs, with high biosecurity levels, the best genetic offer and optimal quality controls that assure a good product in farm. Quality in semen doses is a concept that involves motility, abnormal cells, absence of diseases and contaminants, and genetics. Presence of bacterial contamination in the semen dose has adverse effects, depending on the bacteria present and its concentration [1,2,3,4,5]. It has been evidenced that a ratio between sperm cells and bacteria from 1:1 to 100:1 negatively affects semen. The adverse effects of bacteria on sperm cells are agglutination and chemical variations on the cells environment (ph, osmolarity, conductivity, presence of nocive metabolites), leading sperm cells to dead. Moreover, bacteria can cause fertility problems in sows, such as endometritis, cyclic or acyclic returns to oestrus, and vaginal discharges. These effects become more evident whenever the AI is performed after ovulation, when estrogen concentration is decreasing, or when semen is placed with postcervical or intrauterine insemination [14]. Besides primary pathogens of genitourinary tract, semen obtained from a wealthy mature boar is sterile. Nevertheless, ejaculates are easily contaminated during the process of collecting and its manipulation to the final insemination dose. Contamination can have different sources, as animal or human sources (skin, manure, preputial fluids, respiratory secretions), and environmental sources (air, floor, water, desks, materials). To minimize risks and enhance non contaminated semen, antimicrobials are placed in extenders, but also a strict hygienic protocol should be put in place in AI boar studs, based on critical points mentioned [9]. The aim of this study is to identify bacteria presence in semen that showed decreased longevity, and its antibiotic resistance, and enhance the incidence of Serratia species whenever low longevity and bacteria are detected in a semen dose. 2. Materials and Methods The study was performed in six AI boar stations of AIM Ibérica in Spain (five) and Portugal (one). Boars used were terminal boars and pure breed boars. Collection was performed using the gloved hand method. Only sperm rich fraction was collected, then leaded to the laboratory, and extended (1:1, v/v) in a commercial extender. The routine quality control in all the six AI boar stations included longevity check, bacteria screening, identification and eventually, antibiotic resistance check. This study includes results from February 2007 to May One extended sample of each ejaculate obtained every day in the production line was routinely analyzed at hours for motility and bacteria growth. Longevity was analyzed by an expertise technician. Samples were pre-warmed at 34º C during minutes, and then subjective motility was evaluated using a phase contrast microscope. Whenever a sample had less than 50% of motility, the boar was considered out of production. For all ejaculates, an extended semen sample was also analyzed for bacteria growth in blood agar plates, using sterile swabs. Blood agar plates were placed in a warm cabinet at 34-35ºC. The cultures were done h after production. When a

3 blood agar plate showed bacteria growth within 48 h of incubation, and simultaneously the longevity of sperm cells were below 50% at 72-96h, the blood agar plate with the colonies was sent to a microbiological laboratory for bacteria identification. Besides the previous protocol, daily ejaculate cultures were performed for bacteria growth. Raw ejaculates were diluted approximately 1:200 in physiological saline solution (PSS) without extender, and 0.3 ml of the dilution placed in a blood agar plate. The culture was performed using a warm cabinet at 34-35ºC during 48 h. Randomly, blood agar plates with bacteria colonies were sent to a microbiological laboratory for bacteria identification. In both cases, bacteria positive samples of extended semen or raw ejaculates were randomly analyzed for antibiotic resistance. 3. Bacteria population in AI boar stations. Bacteria occurrence in boar stations is unavoidable. Semen obtained from healthy mature boars is sterile, but during the process of collection, dilution and bottling, it is very difficult to avoid bacteria presence in the final product. Nevertheless, bacteria introduced in semen ejaculates can be primary pathogens for boars and sows, or environmental bacteria. The majority of contaminants are environmental bacteria, most of them originating from the family Enterobacteriaceae, although there is great variation of contaminants among AI boar stations and countries. A few previous studies report different bacteria occurrence in porcine undiluted raw semen [6,8] and ejaculates [7]. In order to contain these microorganisms, commercial extenders are added with antibiotics that mostly remove the undesirable bacteria from the sperm doses. A retrospective study of bacteriology in six AI boar stations was made on semen that had bacteria growth and showed decreased motility at h after production. Bacteria is proven to decrease sperm longevity, and cause sperm agglutination, besides reproductive problems in inseminated sows. It has been found single or multiple bacteria growth in sperm doses [7]. In our study (n=128), a total of 114 samples presented only one bacterial strain, whereas 14 samples presented two bacterial strains. In table 1 it is shown the incidence of bacteria for all samples. For four samples, identification was not possible. Serratia liquefaciens was present the 26,1%. Moreover, Serratia strains were present in the 44,44% of the samples (Serratia liquefaciens, 26,1%; Serratia marcescens, 16,3%; Serratia odorifera, 3,5%, Serratia spp., 3,5%). All isolated analyzed bacteria were found to be resistant to Beta-lactamics ampicillin and amoxicillin and the cephalosporine cefuroxime. Most of them showed resistance to the aminoglycosides expectinomicine and neomicyn, and to the Beta-lactamic amoxicillinclavulanic. Serratia is a ubiquitous Gram negative facultatively anaerobic microorganism, in the family Enterobacteriaceae. The family Serratia have ten species, S. entomophila, S. ficaria, S. fonticola, S. grimesii, S. liquefaciens, S. proteamaculans (with two subspecies, Proteamaculans and Quinovora), S. marcescens, S. odorifera, S. plymuthica and S. rubidae. Serratia grows slowly in blood agar plates when compared to other Enterobacteriaceae. Besides, the colonies are round, but may differ in thickness,

4 hemolytic ability, and color. This make difficult the isolation and early identification of Serratia in the AI boar station. 4. Antibiotics and resistance in vitro Antibiotics used for antibiogram in the present study were those suggested by the microbiological laboratory. Althouse et al. (6) found that bacteria isolated from extended porcine semen exhibiting poor sperm longevity had low antibiotic sensitivity. Antibiotics recommended for boar sperm extenders are included in the 90/429/CEE Directive. Effective antibiotics against leptospirosis and mycoplasmosis are required: 500 IU of streptomycin per ml, or 500 IU of penicillin per ml, or 150 µg of lincomycin per ml, or 300 µg of spectinomycin per ml, or equivalents. Nevertheless, commercial extenders are added with a high variety of antibiotics. The most common antimicrobials used in porcine semen extenders are Aminocyclitols (Spectinomycin); Aminoglycosides (Amakacin, Gentamicin, Neomycin, Streptomycin); β-lactams (Amoxicillin, Penicillin, Ticarcillin); Lincosamides (Lincomycin); Macrolides (Tylosin), Polypeptides (Polymixin); Quinolones (Enrofloxacin, Norfloxacin) [9], and combinations of them. Sometimes, these combinations generate interactions among antibiotics. Certain β- lactams can interact with aminoglycosides and lead to inactivation of both antimicrobials [10,11]. It has been also reported degradation of gentamicin in dextrose solutions [12]. In the present study, all bacteria isolated were found to be in vitro resistant to the β- lactams amoxicillin and ampicilline and to the cephalosporine cefuroxime. Moreover, above 90% of the colonies were also resistant to other common antibiotics present in commercial extenders, such as the aminoglycosides neomicyn and espectinomycin, and the β-lactam amoxicilin-clavulanic acid. These findings are according to those obtained by Althouse et al. (6) in a previous research. Antibiotic resistance found in this study is presented in table 2. The huge and uncontrolled use of antibiotics in porcine semen extenders encourages the induction of bacterial adaptive or associated resistances. Correct use, identification and dosage of antibiotics present in commercial extenders should be a goal for sustainability and effectiveness of semen extender production in the future. The antibiotics contained in the extender used in all AI boar studs of the study were neomicyn and penicillin. Whenever semen tenability was endangered and contamination was not controlled, enrofloxacine was added to the extender to remove bacteria from the doses, and get some time to look for the contamination source. It was a surprise that all contamination was removed in 123 out of 128 cases. Enrofloxacine proved to be effective in vitro in 52,1% of the samples, and proved to be ineffective in vitro in 45,8% of the samples (table 2). Whenever enrofloxacine was unable to remove contamination in a short term, ceftiofur was successfully applied. Bacteria resistant to enrofloxacine but sensible to ceftiofur were Pantoea spp. in two cases, Klebsiella spp. in two cases, and Pseudomona oryzhabitans in one case. 5. Conclusions

5 The complete elimination of bacteria in the AI boar station is not a feasible goal. Nevertheless, a significant and sufficient reduction of bacterial load by improving collection hygiene, personal hygiene during production, and good regular monitoring form bacterial contamination in critical points is attainable [6]. A Prudent use of antibiotics in semen extenders is essential for good quality and longevity of semen, as long as its use is rational and controlled. Administration of extra antibiotics should be done only when all options are exhausted, and for a limited time. All quality controls done on a sperm dose are retrospective controls. Whenever the result is not good, the dose has already been used. For that reason, we should be able to advance the problems, and shorten our reaction time. Monitoring extended semen samples is essential for bacteriological control, but also other critical points concerning production, such as distillated water tanks, desks, materials, walls, raw semen, and many others should be analyzed. Every individual station should identify and develop a custom monitoring program for bacteria control. In this retrospective study, almost half of the isolations performed when longevity of sperm cells was low, presented Serratia as primary semen contaminant. Although this bacteria not always was sensible to enrofloxacine, always disappeared from sperm doses when applied. Sensibility in vitro may be different to in vivo, as other factors may influence the results, like combination of antimicrobials, ph, or temperature. Further research should be done to investigate the effect of Serratia species on sperm doses, and how this bacterium interacts with other environmental bacteria present. 6. Bibliography 1.- Teague NS, Bogarsky S, Glenn JF, 1971: Interference of human spermatozoa motility by Escherichia coli. Fertil Steril 22, Auroux MR, Jacques L, Mathieu D, Auer J, 1991: Is the sperm bacterial ratio a determining factor in impairment of sperm motility: an in-vitro study in man with Escherichia coli. Int J Androl 14, Wolf H, Panhans A, Stolz W, Meurer M, 1993: Adherence of Escherichia coli to sperm: a mannose mediated phenomenon leading to agglutination of sperm and E. coli. Fertil Steril 60, Monga M, Roberts JA, 1994: Sperm agglutination by bacteria: receptor-specific interactions. J Androl 15, Diemer T, Weidner W, Michelmann HW, Schiefer HG, Rovan E, Mayer F, 1996: Influence of Escherichia coli on motility parameters of human spermatozoa in vitro. Int J Androl 19, Althouse GC, Kuster CE, Clark SG, Weisiger RM, 2000: Field investigations of bacterial contaminants and their effects on extended porcine semen. Theriogenology 53, Althouse GC, Lu KG, 2005: Bacteriospermia in extended porcine semen. Theriogenology 63, Althouse GC, Pierdon MS, Lu KG, 2008: Thermotemporal dynamics of contaminant bacteria and antimicrobials in extended porcine semen. Theriogenology 70,

6 9.- Althouse GC, Sanitary procedures for the production of extended semen. Reprod Dom Anim 43 (suppl. 2), Perényi T, Graber H, Arr M. Über die Wechselwirkung der Penizilline und Aminoglykosid-Antibiotika. Int J Clin Pharmacol Ther Toxicol 1974; 10: Farchione LA. Inactivation of aminoglycosides by penicillins. J Antimicrob Chemother 1981; 8(Suppl. A): Graham AE, Speicher E, Williamson B. Analysis of gentamicin sulfate and a study of its degradation in dextrose solution. J Pharm Biomed Anal 1997; 15: Tamuli MK, Sharma DK, Rajkonwar CK. Studies on the microbial flora of boar semen. Indian Vet J 1984; 61: Martínez EA, Vázquez JM, Roca J, Lucas X, Gil MA, Parrilla I, et al. Successful non-surgical intrauterine insemination with low number of spermatozoa in sows by a fiberoptic endocope technique. Reproduction 2001; 122: Grimont F and Grimont PAD Genus XXXIV Serratia in Bergey s manual of Systematic Bacteriologi 2 nd Ed. Vol 2B: Springer, Table 1 Bacteria isolated from contaminated samples, in which sperm cells showed decreased motility at 72-96h. Samples were taken and analyzed during a 3 years period, between 2007 and 2010 BACTERIA ISOLATED FREQUENCY OF ISOLATIONS (No.) PERCENTAGE n=142 Serratia liquefaciens 37 26,1% Serratia marcescens 17 12,0% Staphylococcus spp 8 5,6% Microccocus spp 8 5,6% Pantoea spp 7 4,9% Pseudomonas spp 7 4,9% Klebsiella spp 6 4,2% E. Coli 5 3,5% Serratia spp. 5 3,5% Serratia odorifera 5 3,5% Stenotrophomonas maltophila 5 3,5% Pseudomonas aeruginosa 5 3,5% Not identified bacteria 4 2,8% Pseudomonas oryzihabitans 3 2,1% Streptococcus spp 3 2,1% Aeromonas hydrophila 2 1,4% Pseudomonas luteola 2 1,4% Providencia 2 1,4% Enterobacter asburiae 1 0,7% Actinobacillus Ureae 1 0,7% Pandoraea spp 1 0,7% Myroides species 1 0,7% Pseudomonas stutzeri 1 0,7% Bacillus asacarolíticos 1 0,7% Bacillus spp 1 0,7% Proteus spp 1 0,7% Acinetobacter wolfii 1 0,7% Enterobacter fecalis 1 0,7% Pasteurella aerogenes 1 0,7%

7 Table 2 Resistance of diverse bacteria found in 128 analysis of Spanish and Portuguese boar studs with decreased longevity sperm cells and bacteria growth at 72-96h R I S n β-lactam Ampicillin 100,0% 0,0% 0,0% 60 β-lactam Amoxicilin 100,0% 0,0% 0,0% 51 Cephalosporine Cefuroxime 100,0% 0,0% 0,0% 9 Aminoglycoside Espectinomycine 98,0% 0,0% 2,0% 51 β-lactam Amoxicillin+Clavulanic 96,7% 3,3% 0,0% 60 Aminoglycoside Neomicyn 96,1% 2,0% 2,0% 51 Aminoglycoside-lincosamide Lincospectin (espectinomycinelincomycine) 81,8% 0,0% 18,2% 22 Aminoglycoside Gentamicine 80,0% 1,7% 18,3% 60 Tetracicline Oxitetraciclin 66,7% 23,5% 9,8% 51 Sulfonamide+TMP Sulfametoxazol+TMP 58,8% 9,8% 31,4% 51 Fluoroquinolone Marbofloxacine 57,1% 28,6% 14,3% 7 Fluoroquinolone Enrofloxacine 52,1% 2,1% 45,8% 48 Aminoglycoside Apramicine 43,1% 33,3% 23,5% 51 Polymyxin Colistine 41,2% 5,9% 52,9% 51 Fluoroquinolone Ciprofloxacine 28,6% 14,3% 57,1% 7 Cephalosporine Cefoxitin 11,1% 0,0% 88,9% 9 Cephalosporine Cefotaxime 11,1% 0,0% 89,9% 9 Cephalosporine Ceftazidime 11,1% 0,0% 89,9% 9 Cephalosporine Cefepime 11,1% 0,0% 89,9% 9 Aminoglycoside Amilkacine 11,1% 0,0% 89,9% 9 Cephalosporine Ceftiofur 9,8% 3,9% 86,3% 51 R= Bacteria showed resistance to the antibiotic I= Bacteria showed intermediate resistance to the antibiotic S= Bacteria was sensible to the antibiotic