ÞEMËS ÛKIO MOKSLAI. 2004. Nr. 2. P. 44–51 Modestas Ruþauskas, Èeslova Butrimaitë-Ambrozevièienë Lietuvos mokslø akademijos leidykla, 2004 Most common pathogenic bacteria and their Pathological material from pigs was investigated in the Veterinary Ins- titute of Lithuanian Veterinary Academy and National Veterinary Labo- ratory during a 5-year period. Pathological material was investigated under suspicion of infections caused by pathogenic bacteria with the aim to isolate them and to determine their resistance to antimicrobials. In Lithuanian pig farms, among pathogenic and opportunistic bacteria most frequently E. coli and Salmonella spp. 176 strains of E. coli and 122 strains of Salmonella were isolated. The most common serotypes of E.
coli were 08:K87, 0141:K85, and 0149:K91. Most of them contained fimbrial antigens F4, F5 or F6. S. Choleraesuis (79%) was the most common serovar among Salmonella. The highly pathogenic S. Enteriti- dis and S. Typhimurium were infrequent (respectively 3.3% and 2.5%).
The HAP group (Haemophilus, Actinobacillus and Pasteurella) bacteria were also rather widespread: 84 strains of this group were isolated. The most frequent pathogens were Actinobacillus pleuropneumoniae, Haemo- philus parasuis and Pasteurella multocida.
The bacteria isolated from pigs were comparatively resistant to an- timicrobials. E. coli and Salmonella spp. were most susceptible to poly- myxin B and fluoroquinolones. The resistance to other antimicrobials was higher, especially to lincomycin-spectinomycin and aminoglycosides.
25% of staphylococci were resistant to penicillin, and 50% of strep- tococci were resistant to gentamicin. Bacteria of the HAP group were susceptible to many antimicrobials. All investigated Pasteurella multocida were tetracycline-sensitive, whereas Haemophilus and Actinobacillus spp.
were susceptible to chloramphenicol, ceftiofur and polymyxin B.
Key words: antimicrobials, E. coli, HAP, resistance, Salmonella teria occupy one of the most important places among all pathogens, particularly by their adaptability and Pathogenic bacteria are important in the etiology of ability to resist unfavourable environment.
pig diseases and cause great economical losses [18].
Resistance of bacteria to antimicrobials has re- Bacteria can cause diseases by themselves or in as- cently become a question of topical importance. Re- sociation with viruses, mycoplasmas or other micro- search works on this subject appear all over the organisms. Some studies of infectious diseases in world. Some works put emphasis on separate spe- Lithuanian pig farms were carried out. Species of cies or genera [6, 21, 26, 30] of bacteria, others pathogenic bacteria, mycoplasmas, chlamydias or vi- concentrate on the resistance of bacteria to separa- ruses were investigated [9, 11, 16, 24, 28, 29]. Bac- te antibiotics or their groups [13, 25]. Of great im- Most common pathogenic bacteria and their antimicrobial resistance in pigs portance are studies on the sensitivity of pathogenic For inoculation into media, 0.5 McFarland units of bacteria in different regions or countries [3]. Sym- bacterial suspension (for Haemophilus and Strepto- posia and conferences devoted to the theme of re- coccus 1 McFarland unit) were used. The pH of sistance to antibiotics are held [27, 31]. Lithuania media was 7.4 ± 0.2. Antibiotic discs (Becton Dic- has not so far developed an apparent strategy to kinson, USA) were used and the results were inter- ensure the prudent use of antibiotics for veterinary preted with special tables of this manufacturer. The purposes. The aim of the present work was to elu- concentrations of antimicrobials were standard: pe- cidate the most widespread pathogenic bacteria in nicillin 10 U, oxacillin 1 µg, chloramphenicol 30 µg, Lithuanian pig farms and determine their resistance enrofloxacin 10 µg, gentamicin 10 µg, polymyxin B 300 U, streptomycin 10 µg, tetracycline 30 µg, cef- tiofur 30 µg. Resistance of the isolated bacteria to The pathological material of pigs was provided from different Lithuanian districts. Investigations were car- ried out at the Veterinary Institute of Lithuanian Investigations involved 35 pig farms from all regions Veterinary Academy and in National Veterinary La- of Lithuania. Data listed in Table 1 reveal the most common pathogenic bacteria in pigs. E. coli was Bacteriological investigations were done using the most widespread pathogenic bacteria in Lithua- classical methods. The common media for isolation nian pig farms: 176 E. coli isolates were obtained of bacteria were used: Tryptone Soy Agar, Tryptone (19%) from 925 tested pigs, and 156 strains of iso- Soy Broth, Nutrient Agar (Oxoid, Lab-M, England) lated E. coli were serotyped; 20 strains did not ag- and analogues. For cultivation of different bacteria, glutinate with agglutinating sera. According to the selective media were used: Endo Agar, SS Agar, data of Table 2, the most widespread serotypes of Perfringens Agar Base (Oxoid, Lab-M, England), as E. coli were 0141:K85, 08:K87 and 0149:K91. Most well as adequate ingredients and needful supple- of ETEC (enterotoxigenic) E. coli contained fimb- ments. For cultivation of fastidious bacteria, growth rial antigens F4, F5 or F6. E. coli of serotype 0157 factors were used (Nicotinamide Adenine Dinucleo- tide (Sigma)). For isolation of staphylococci Manite Salt Agar (Oxoid, England), Baird Parker Medium Table 1. Data on bacteria isolated from pigs (Lab M, England) and of streptococci Columbia Broth (BBL, USA), Eugon Agar (bio Merieux, Fran- ce) were used. E. coli were incubated at 43 °C with the aim to inhibit the growth of saprophytic E. coli.
Identification of isolated bacteria was done according to their growth, biochemical properties, according to Bergey’s Manual of Determinative Bacteriology [2]. Some experiments were based on standard met- hods, e.g., ISO standard for isolation of Salmonella (ISO 6579–1993). Bacteria difficult to cultivate on media (Brachyspira hyodysenteriae) were examined For determination of biochemical properties, car- bohydrates and amino acids in test tubes with indi- cators, as well as the commercial systems Intersis- Table 2. Prevalence of Salmonella serovars in pigs tem (Liofilchem, Italy) and Crystal (BBL, USA) were used. Escherichia coli and Salmonella were serotyped by drop on glass or latex agglutination test with commercial sera. For Salmonella typing, SIFIN (Ger- many) and for E. coli the Sanofi Diagnostics Pa- steur (France), Oxoid (England) and Bundesinstitut Veterinärmed (Germany) sera were used.
The resistance to antimicrobials was determined by the method of diffusion in agar gel according to Kirby–Bauer [1]. Mueller Hinton Agar II and Hae- mophilus Test Medium (Oxoid, England) were used.
Modestas Ruþauskas, Èeslova Butrimaitë-Ambrozevièienë has not so far been isolated in Lithuania. These countries about salmonella serovars’ prevalence va- serotypes are also widespread in all countries and ry. There is a great variety of salmonella serotypes our data coincide with the data of other authors in some countries, particularly in the U.S.A [5]. At present, more than 2500 Salmonella serovars are There are different E. coli categories associated known. Most of them are extremely rare. Only a with animal colibacillosis such as enterotoxigenic, ve- few serovars (e.g., Enteritidis, Typhimurium, Chole- rotoxigenic, attaching and effacing, septicemic, uropa- raesuis, Hadar, etc.) are distributed worldwide and thogenic. Enterotoxigenic E. coli is the most common are important from the epidemiological point of category in pigs. This category includes serotypes that affect newborn piglets and cause extraintestinal dise- Salmonella Choleraesuis preferentially infects pigs ase in preweaned and weaned pigs, manifested clini- and can cause increased morbidity and mortality re- cally by sudden death and pathologically by lesions sulting in millions of lost income for the pork in- caused by endotoxic shock. These strains most often dustry. This serovar is more common in Eastern belong to serotypes O8, O149, and O157 and are gene Europe. However, in West European countries Sal- probe positive for haemolysin, K88 (F4) fimbriae, he- monella Typhimurium replaced this serovar.
at-labile enterotoxin I (LT-I), and heat-stable entero- Salmonella and E. coli can be mentioned as the toxin b (STb) [10]. Most of E. coli isolated by us con- most common agents of digestive tract infections tained these antigens, particularly K88 (F4). Veroto- [15], although the primarily intestinal bacteria, Sal- xigenic E. coli was associated with oedemal disease in monella and Escherichia coli, are widespread in the environment and commonly found in farm effluents, In the last years, agents of pig salmonellosis we- sewage and in any material subject to faecal conta- re also rather widespread. 122 strains of Salmonella mination. Salmonellosis and colibacillosis have been (9.9%) were isolated from 1237 pigs. So far, the recognized in all countries, but they appear to be variety of Salmonella serovars in Lithuanian farms most prevalent in areas of intensive animal hus- has been small. The most widespread serovar was bandry, especially poultry or pigs and dairy cattle S. Choleraesuis (79%) (Table 3). The genus Salmo- reared in confinement. Salmonellosis and colibacil- nella consists of two species: S. enterica and S. bon- losis can affect all species of domestic animals; young gori. Species names were arbitrarily given to sero- animals, pregnant and lactating animals are those vars for convenience reasons in medical practice.
most susceptible. Some serotypes only affect certain The serovar S. Choleraesuis depends to the species hosts, e.g., S. Choleraesuis or E. coli O149:K91 in Salmonella enterica subsp. enterica. Analogous data pigs, although most serotypes may cause a disease are presented also by authors from the neighbou- in a wide range of animal species. Enteric disease ring countries. For example, in Poland in 2001 19 is the commonest clinical manifestation, but a wide isolates from 33 depended to the serovar Salmonel- range of clinical signs that include acute septicae- la Choleraesuis [14]. In recent years, new and by mia, abortion, arthritis and respiratory disease may far more variable Salmonella serovars have been re- be seen. Many animals, however, may also be in- gistered in Lithuania. This may be accounted for by fected but show no clinical illness. Such animals may an increasing flow of animal products and fodder be important in relation to the spread of infection imported from other countries. Data from other among the herds and as causes of human food Bacteria of the HAP group are rather widespread Table 3. Serotypes of E. coli isolated from pigs in Lithuanian pig farms. Fifty Haemophilus and Acti- nobacillus isolates were obtained (19.5%) from 256 pigs. A. pleuropneumoniae and H. parasuis are equally widespread, P. multocida is more infrequent (34 strains (8.6%) were isolated). P. multocida in most cases acts as the secondary agent. Only in one case highly viru- lent Pasteurella was isolated from the blood.
Forty-five strains of Staphylococcus and the sa- me number of Streptococcus were isolated (15.6% and 19.6%, respectively). The most widespread among staphylococcis (pathogenic in pigs) were S.
hyicus and S. aureus. The isolated streptococci be- Among clostridia the most frequent was C. per- Most common pathogenic bacteria and their antimicrobial resistance in pigs Table 4. E. coli resistance to antimicrobials (n = 55) of strains Susceptible % Resistant % Intermediate % Table 5. Salmonella resistance to antimicrobials (n = 28) of strains Susceptible % Resistant % Intermediate % Table 6. Pasteurella multocida resistance to antimicrobials (n = 12) of strains Susceptible % Resistant % Intermediate % These bacteria demonstrated a 100% susceptibility in Table 9 contains data on the resistance of Strep- vitro to ceftiofur, polymyxin B and chloramphenicol.
tococcus to antimicrobials. No streptococci resistant Of all staphylococci, 75% were susceptible to pe- to penicillin were isolated, but 29.4% of them were nicillin (Table 8). However, even one fourth of them medium susceptible; 29.2% were resistant to neomy- were penicillin-G resistant. All staphylococci were cin and even half of the strains were resistant to susceptible or medium susceptible to aminoglycosi- gentamicin and lincomycin-spectinomycin. Faecal des except streptomycin (25% resistant).
streptococci demonstrated the highest resistance.
Modestas Ruþauskas, Èeslova Butrimaitë-Ambrozevièienë Table 7. Actinobacillus–Haemophilus resistance to antimicrobials (n = 13) of strains Susceptible % Resistant % Intermediate % Table 8. Staphylococcus resistance to antimicrobials (n = 11) of strains Susceptible % Resistant % Intermediate % Table 9. Streptococcus resistance to antimicrobials (n = 31) of strains Susceptible % Resistant % Intermediate % Among pathogenic bacteria able to cause disea- PCR have been introduced in Lithuania not long ses in pigs, E. coli and Salmonella spp. are belong ago, there are no generalized data on some bacte- to those most widespread in Lithuania. The seroty- rial diseases caused by the agents that are difficult pe variety of these bacteria is not great. However, to cultivate on nutrient media. However, judging the developing trade relations with other countries from clinical manifestations, the course of disease, contribute to the increasing variability of these sero- pathological anatomical changes and results of sero- types. No such dangerous bacteria as toxigenic E.
logical tests we may assume that in Lithuanian pig Most common pathogenic bacteria and their antimicrobial resistance in pigs farms the cases of leptospirosis, swine dysentery, pro- tible to many antimicrobials used in veterinary prac- liferative ileitis and mycoplasmosis are not infrequ- ent. Some bacterial diseases occur only in separate 3. Some pathogenic bacteria, particularly Salmo- pig-breeding farms as a result of vaccination (erysi- nella spp. and E. coli, tend to be more resistant to pelas). These diseases are important in pig-breeding Bacteria isolated from pigs are comparatively resistant to antimicrobials. Enterobacteriaceae (Es- cherichia coli and Salmonella spp.) were most su- sceptible to polymyxins and fluoroquinolones. They showed an increasing resistance to other antimic- 1. Bauer A. W., Kirby W. M., Serris J. C., et al. Anti- robials such as lincomycin-spectinomycin and amino- biotic susceptibility by a standardized single disc // glycosides in particular. Bacteria of the HAP group American Journal of Clinical Pathology. 1966. Vol. 45.
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tibiotikams. Ketvirtadalis iðskirtø stafilokokø buvo atspa- enterica serovar typhimurium from humans and pro- rûs penicilinui, o net pusë tirtø streptokokø – gentamici- duction animals // Journal Antimicrobial Chemotera- nui. HAP grupës bakterijos buvo gerokai jautresnës ávai- riø grupiø antimikrobinëms medþiagoms.
27. Stuart B. L. Antibiotic resistance: an ecological imba- Raktaþodþiai: antimikrobinës medþiagos, E. coli, HAP, lance // Symposium on Antibiotic Resistance: origins, Most common pathogenic bacteria and their antimicrobial resistance in pigs ñåðîòèïû O141:K85, O149:K91 è O8:K87. ×àñòî îíè ×åñëîâà Áóòðèìàéòå-Àìáðîçÿâè÷åíå èìåëè àäãåçèîííûå àíòèãåíû F4, F5 èëè F6. Òàêæå ÁÎËÅÅ ÐÀÑÏÐÅÄÅËÅÍÍÛÅ ÏÀÒÎÃÅÍÍÛÅ ÷àñòî âûäåëÿëè ñàëüìîíåëëû. Ñàìûì ðàñïðîñòðà- ÁÀÊÒÅÐÈÈ ÑÂÈÍÅÉ È ÈÕ ÓÑÒÎÉ×ÈÂÎÑÒÜ íåííûì ñåðîâàðîì ñàëüìîíåëë áûë ñåðîâàð S. Cho- Ê ÀÍÒÈÌÈÊÐÎÁÍÛÌ ÂÅÙÅÑÒÂÀÌ leraesuis (79%). Ïàòîãåííûõ S. Enteritidis è S. Typhi-murium âûäåëåíî íàìíîãî ðåæå (ñîîòâåòñòâåííî 3,3 è 2,5%). Äîâîëüíî ðàñïðîñòðàíåíû áûëè òàêæå áàê- Áàêòåðèîëîãè÷åñêèå èññëåäîâàíèÿ ïàòîëîãè÷åñêîãî òåðèè ãðóïïû HAP (Haemophilus, Actinobacillus è Pa- ìàòåðèàëà ñâèíåé ïðîâîäèëèñü â Èíñòèòóòå âåòåðè- steurella). Âûäåëåíû 84 èçîëÿòà ýòîé ãðóïïû, íàèáî- íàðèè Ëèòîâñêîé âåòåðèíàðíîé àêàäåìèè è Íàöèî- ëåå ÷àñòî âèäû Actinobacillus pleuropneumoniae, Hae- íàëüíîé âåòåðèíàðíîé ëàáîðàòîðèè â òå÷åíèå ïÿòè mophilus parasuis è Pasteurella multocida.
ëåò ñ öåëüþ âûäåëèòü ïàòîãåííûå áàêòåðèè è óñòà- Áîëüøèíñòâî âûäåëåííûõ ñàëüìîíåëë è ýøåðè- íîâèòü èõ óñòîé÷èâîñòü â îòíîøåíèè àíòèìèêðîá- õèé in vitro áûëè ÷óâñòâèòåëüíû ê ïîëèìèêñèíàì è íûõ âåùåñòâ. Èññëåäîâàíèÿ ïðîâîäèëèñü âî âðåìÿ ôòîðîõèíîëîíàì, íî óñòîé÷èâû ê ëèíêîìèöèíó, âñïûøåê çàáîëåâàíèé ñâèíåé ïðè ïîäîçðåíèè, ÷òî ñïåêòèíîìèöèíó, àíòèáèîòèêàì ãðóïïû àìèíîãëè- ýòèîëîãè÷åñêèì ôàêòîðîì áîëåçíåé ÿâëÿþòñÿ ïàòî- êîçèäîâ. Óñòîé÷èâû ê ïåíèöèëëèíó áûëè 25% ñòà- ãåííûå èëè óñëîâíî-ïàòîãåííûå áàêòåðèè. Áàêòåðèî- ôèëîêîêêîâ, à 50% ñòðåïòîêîêêîâ áûëè óñòîé÷èâû ëîãè÷åñêèå âûñåâû îñóùåñòâëÿëèñü èç ïàðåíõèìà- ê ãåíòàìèöèíó. Áàêòåðèè ãðóïïû HAP áûëè áîëåå òîçíûõ îðãàíîâ, à â îòäåëüíûõ ñëó÷àÿõ – èç òåõ ÷óâñòâèòåëüíû ê àíòèìèêðîáíûì âåùåñòâàì ðàçíûõ ìåñò, îòêóäà âûäåëèòü ïðåäïîëàãàåìûå áàêòåðèè íàèáîëåå âåðîÿòíî. Áîëåå âñåãî âûäåëåíî áàêòåðèé Êëþ÷åâûå ñëîâà: àíòèìèêðîáíûå âåùåñòâà, E. co- âèäà E. coli. Èç íèõ ÷àùå âñåãî óñòàíàâëèâàëè li, HAP, óñòîé÷èâîñòü, Salmonella

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