Three studies were conducted to assess if the ability of Enterococcus spp. surviving the ensiling process and bovine gastrointestinal tract could impact risk of bovine mastitis. The first study determined ability of enterococci to survive 3 wk ensiling. Grass and corn crops were divided into 3 treatments: 2 commercial silage inoculants, 1 negative control. After wk 1, 2, and 3 of ensiling, dry matter and bacterial enumeration were performed. Addition of silage inoculant led to greater levels of enterococci in grass silage compared with negative control levels, but showed less difference in inoculated corn silage. The second study quantified enterococci shedding rates in lactating dairy cows. Using a 4 x 4 Latin Square design, lactating, ruminally fistulated Holsteins were inoculated with enterococcal isolates from silage inoculants, ensiled forages, or clinical mastitis cases. Over the 4-period study, each period consisted of rumen and fecal sampling (2 wk) followed by a wash period (10 d). There were no significant differences in rumen or fecal enterococci levels between the 4 treatments. Both rumen and fecal enterococci levels showed significant differences between baseline and treatment periods (period 3, 4). The third study analyzed similarity in enterococcal isolates of silage and bovine origin using pulsed-field gel electrophoresis patterns from SmaI restrictions. Dendogram analysis showed none of the isolates met or were greater than a 75% genetic similarity and produced a genetically diverse population. Results suggest Enterococcus spp. from silage inoculants are not likely to contribute to an increased risk of enterococcal bovine mastitis. / Master of Science

Multiresistenta bakterier så som meticillinresistenta Staphylococcus aureus (MRSA), bakterier som producerar extended-spectrum beta-lactamase (ESBL) och vancomycinresistenta enterokocker (VRE) är ett problem sedan årtionden tillbaka och som ökar för varje år. Idag på mikrobiologen, Unilabs Skövde, isoleras bakteriestammar från selektiva medier för just MRSA, ESBL och VRE på blodagar innan resistensbestämningarna utförs. Syftet med studien var därför att undersöka möjligheten att göra diskdiffusionstest direkt från de selektiva medierna och således kunna svara ut resultaten tidigare. Utvärdering av detta gjordes genom att undersöka om storleken på antibiotikazonerna för sammanlagt 64 isolat påverkades av att bakterierna som användes vuxit på ett selektivt agarmedium i förhållande till om de vuxit på blodagar. Resultatet visade vad som ansågs vara en normal variation på maximalt ±2 mm för alla parvisa zoner utom en på 3 mm. Av alla zoner som undersöktes för MRSA, ESBL och VRE var majoriteten identiska i antal millimeter, 62 %, 89 % och 98 % respektive. Baserat på det goda resultatet ansågs materialet vara tillräckligt stort för att göra bedömningen att metoden är utförbar. Med tanke på de positiva effekterna av att göra resistensbestämningar direkt från de selektiva agarmedierna görs rekommendationen till mikrobiologen, Unilabs Skövde, att övergå till denna metod. / Multiresistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum beta-lactamase (ESBL) producing bacteria and vancomycin-resistant enterococci (VRE) have been a problem for decades with an increasing rate. Today, at mikrobiologen, Unilabs Skövde, bacterial strains are isolated from selective media for MRSA, ESBL and VRE onto blood agar before the susceptibility testing. The aim of the study was to examine the possibility of disk diffusion susceptibility testing directly from the selective media and thus be able to reply the findings earlier. The zones of inhibition were examined for a total of 64 isolates after disk diffusion testing from both the selective and blood agar plates in order to evaluate if the zone sizes were affected. The results showed what was considered a normal variation of ±2 mm for all pairwise zones except for a difference in 3 mm. The majority of all zones tested for MRSA, ESBL and VRE had equally large zones, 62%, 89% and 98% respectively. Based on the good results, the material was considered enough to make the conclusion that the method is feasible. Considering the positive effects of making susceptibility testing directly from selective agar, a change to this method is recommended to mikrobiologen, Unilabs Skövde.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Enterococcus resistentes à vancomicina (VRE) são reconhecidos como importantes patógenos causadores de infecções nosocomiais, configurando um grave problema de saúde pública, principalmente pela escassez de opção terapêutica eficaz. O fenótipo de resistência VanA é o mais frequente, sendo definido pela resistência a altos níveis de vancomicina e teicoplanina. VanA é caracterizado por um conjunto gênico (vanRSHAXYZ) localizado no elemento genético móvel denominado transposon Tn1546. A diversidade de Tn1546 resulta de alterações estruturais promovidas por deleções ou integração de sequências de inserção (IS) que, exercem papel chave na evolução do elemento VanA, modificando os aspectos relacionados à sua transferência e expressão do fenótipo. O objetivo deste estudo foi caracterizar e avaliar o polimorfismo de elementos Tn1546 presentes em amostras de diferentes espécies de Enterococcus isoladas em instituições hospitalares do Estado do Rio de Janeiro no período de 2000 a 2012. Foram incluídas neste estudo 70 amostras VRE que foram caracterizadas quanto ao gênero, espécies e genótipo de resistência aos glicopeptídeos por métodos convencionais e PCR multiplex. A susceptibilidade a 17 antimicrobianos foi avaliada pelo método de difusão em ágar, e concentração inibitória mínima (CIM) para vancomicina e teicoplanina foi determinada por microdiluição em caldo. O tranposon foi obtido após lise das células bacterianas e amplificação por PCR longo, utilizando-se oligonucleotídeos específicos para a região repetida e invertida que flanqueia este elemento genético. A diversidade dos elementos Tn1546 foi avaliada por um conjunto de métodos moleculares que incluiu a análise do polimorfismo do tamanho de fragmentos de restrição (restriction fragment lenght polymorphism, RFLP), utilizando-se a endonuclease ClaI, amplificação de segmentos internos por PCR de sobreposição de oligonucleotídeos (overlapping PCR) e detecção de sequências de inserção (ISs). A caracterização em espécies considerada para as demais análises foi obtida pela metodologia de PCR de acordo com a seguinte distribuição: E. avium (N=6), E. faecalis (N=12), E. faecium (N=46), E. gallinarum (N=4) e E. raffinosus (N=2). Todas as amostras apresentaram o genótipo vanA. Nos testes de susceptibilidade aos antimicrobianos foi observado que todas as amostras foram multirresistentes, sendo resistente de 6 a 13 dentre os 17 antimicrobianos testados. A presença de elementos semelhantes ao arquétipo de Tn1546 foi observada em 61,5% das amostras; entretanto, 27 amostras apresentaram perfis variantes de Tn1546. Foram identificados nove perfis de RFLP, dentre 66 avaliadas, sendo o perfil I, prevalente e semelhante ao arquétipo de Tn1546. Não foi possível analisar quatro amostras por RFLP. Os produtos de amplificação de Tn1546 alterados, obtidos pela overlapping PCR e pelo rastreamento de IS, levaram à classificação de 15 tipos polimórficos, nomeados de A a O. A maioria dos Tn1546 polimórficos teve suas regiões de ORF1 e/ou ORF2 deletadas; e IS1542 juntamente com IS1216V foram as inserções mais frequentes, que em muitas situações compartilhavam a mesma região de inserção. IS19 foi detectada apenas na região vanS-vanH. Os dados apresentados neste estudo indicam que o polimorfismo de Tn1546 pode ser explorado no rastreamento de rotas de transmissão, acompanhamento da dispersão de elementos VanA e investigação da evolução de amostras VRE. / Vancomycin-resistant Enterococcus (VRE) is a leading cause of nosocomial infections, remaining as a public health concern in the last two decades. VanA phenotype is the most frequently encountered and it is responsible for high-level vancomycin and teicoplanin resistance. VanA is characterized by a gene cluster (vanRSHAXYZ) located on the mobile genetic element called Tn1546. The diversity of Tn1546 results from structural changes promoted by deletions or additions of insertion sequences (IS) that play a key role in the evolution of VanA element, changing its transferability and expression of phenotype. The aim of this study was to characterize and evaluate the polymorphism of Tn1546 genetic elements belong to VRE isolates obtained from patients attending in hospitals located in the Rio de Janeiro state, during the period from 2000 to 2012. Seventy VRE strains were included in this study. The strains were identified by conventional physiological testes and multiplex PCR, including the vancomycin resistance phenotype and genotype. Antimicrobial susceptibilities testing were carried out by disk diffusion method for 17 antimicrobials; and the minimal inhibitory concentrations (MIC) values to vancomycin and teicoplanin were evaluated by microdilution technique. Tn1546 were amplified by long-PCR using primers to inverted-repeat sequences flanking the transposon. Tn1546 diversity was evaluated by a set of molecular methods including restriction fragment length polymorphism (RFLP), using the endonuclease ClaI, overlapping PCR and detection of insertion sequence elements (IS). Among the 70 strains, 6 E. avium, 12 E. faecalis, 46 E. faecium, 4 E. gallinarum, and 2 E. raffinosus were characterized by multiplex PCR, as well as the vanA glycopeptide resistance determinant. All the strains were multirresistant, being resistant from 6-13 among the 17 antimicrobials tested. The presence of similar elements to the archetype of Tn1546 was observed in 61.5% of samples; however, 27 samples had variant profiles of Tn1546. Nine RFLP profiles were identified among 66 strains, and the profile I was the most frequent and showed to be similar to the archetype of Tn1546. It was not possible to analyze four strains by RFLP. The amplification products of Tn1546, obtained by overlapping PCR, and screening the IS elements led to the characterization of 15 different types, named A through O. Most Tn1546 had its polymorphisms based on deletion of the ORF1 and / or ORF2 regions; and IS1542 as the most frequent insertion element, which in many cases shared the same region of insertion with IS1216V. IS19 was detected only in vanS-vanH region. The data presented in this study indicate that the polymorphism of Tn1546 can be exploited in tracking transmission routes, monitoring the dispersion of VanA elements and investigation of the evolution of VRE strains.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Enterococcus resistentes à vancomicina (VRE) são reconhecidos como importantes patógenos causadores de infecções nosocomiais, configurando um grave problema de saúde pública, principalmente pela escassez de opção terapêutica eficaz. O fenótipo de resistência VanA é o mais frequente, sendo definido pela resistência a altos níveis de vancomicina e teicoplanina. VanA é caracterizado por um conjunto gênico (vanRSHAXYZ) localizado no elemento genético móvel denominado transposon Tn1546. A diversidade de Tn1546 resulta de alterações estruturais promovidas por deleções ou integração de sequências de inserção (IS) que, exercem papel chave na evolução do elemento VanA, modificando os aspectos relacionados à sua transferência e expressão do fenótipo. O objetivo deste estudo foi caracterizar e avaliar o polimorfismo de elementos Tn1546 presentes em amostras de diferentes espécies de Enterococcus isoladas em instituições hospitalares do Estado do Rio de Janeiro no período de 2000 a 2012. Foram incluídas neste estudo 70 amostras VRE que foram caracterizadas quanto ao gênero, espécies e genótipo de resistência aos glicopeptídeos por métodos convencionais e PCR multiplex. A susceptibilidade a 17 antimicrobianos foi avaliada pelo método de difusão em ágar, e concentração inibitória mínima (CIM) para vancomicina e teicoplanina foi determinada por microdiluição em caldo. O tranposon foi obtido após lise das células bacterianas e amplificação por PCR longo, utilizando-se oligonucleotídeos específicos para a região repetida e invertida que flanqueia este elemento genético. A diversidade dos elementos Tn1546 foi avaliada por um conjunto de métodos moleculares que incluiu a análise do polimorfismo do tamanho de fragmentos de restrição (restriction fragment lenght polymorphism, RFLP), utilizando-se a endonuclease ClaI, amplificação de segmentos internos por PCR de sobreposição de oligonucleotídeos (overlapping PCR) e detecção de sequências de inserção (ISs). A caracterização em espécies considerada para as demais análises foi obtida pela metodologia de PCR de acordo com a seguinte distribuição: E. avium (N=6), E. faecalis (N=12), E. faecium (N=46), E. gallinarum (N=4) e E. raffinosus (N=2). Todas as amostras apresentaram o genótipo vanA. Nos testes de susceptibilidade aos antimicrobianos foi observado que todas as amostras foram multirresistentes, sendo resistente de 6 a 13 dentre os 17 antimicrobianos testados. A presença de elementos semelhantes ao arquétipo de Tn1546 foi observada em 61,5% das amostras; entretanto, 27 amostras apresentaram perfis variantes de Tn1546. Foram identificados nove perfis de RFLP, dentre 66 avaliadas, sendo o perfil I, prevalente e semelhante ao arquétipo de Tn1546. Não foi possível analisar quatro amostras por RFLP. Os produtos de amplificação de Tn1546 alterados, obtidos pela overlapping PCR e pelo rastreamento de IS, levaram à classificação de 15 tipos polimórficos, nomeados de A a O. A maioria dos Tn1546 polimórficos teve suas regiões de ORF1 e/ou ORF2 deletadas; e IS1542 juntamente com IS1216V foram as inserções mais frequentes, que em muitas situações compartilhavam a mesma região de inserção. IS19 foi detectada apenas na região vanS-vanH. Os dados apresentados neste estudo indicam que o polimorfismo de Tn1546 pode ser explorado no rastreamento de rotas de transmissão, acompanhamento da dispersão de elementos VanA e investigação da evolução de amostras VRE. / Vancomycin-resistant Enterococcus (VRE) is a leading cause of nosocomial infections, remaining as a public health concern in the last two decades. VanA phenotype is the most frequently encountered and it is responsible for high-level vancomycin and teicoplanin resistance. VanA is characterized by a gene cluster (vanRSHAXYZ) located on the mobile genetic element called Tn1546. The diversity of Tn1546 results from structural changes promoted by deletions or additions of insertion sequences (IS) that play a key role in the evolution of VanA element, changing its transferability and expression of phenotype. The aim of this study was to characterize and evaluate the polymorphism of Tn1546 genetic elements belong to VRE isolates obtained from patients attending in hospitals located in the Rio de Janeiro state, during the period from 2000 to 2012. Seventy VRE strains were included in this study. The strains were identified by conventional physiological testes and multiplex PCR, including the vancomycin resistance phenotype and genotype. Antimicrobial susceptibilities testing were carried out by disk diffusion method for 17 antimicrobials; and the minimal inhibitory concentrations (MIC) values to vancomycin and teicoplanin were evaluated by microdilution technique. Tn1546 were amplified by long-PCR using primers to inverted-repeat sequences flanking the transposon. Tn1546 diversity was evaluated by a set of molecular methods including restriction fragment length polymorphism (RFLP), using the endonuclease ClaI, overlapping PCR and detection of insertion sequence elements (IS). Among the 70 strains, 6 E. avium, 12 E. faecalis, 46 E. faecium, 4 E. gallinarum, and 2 E. raffinosus were characterized by multiplex PCR, as well as the vanA glycopeptide resistance determinant. All the strains were multirresistant, being resistant from 6-13 among the 17 antimicrobials tested. The presence of similar elements to the archetype of Tn1546 was observed in 61.5% of samples; however, 27 samples had variant profiles of Tn1546. Nine RFLP profiles were identified among 66 strains, and the profile I was the most frequent and showed to be similar to the archetype of Tn1546. It was not possible to analyze four strains by RFLP. The amplification products of Tn1546, obtained by overlapping PCR, and screening the IS elements led to the characterization of 15 different types, named A through O. Most Tn1546 had its polymorphisms based on deletion of the ORF1 and / or ORF2 regions; and IS1542 as the most frequent insertion element, which in many cases shared the same region of insertion with IS1216V. IS19 was detected only in vanS-vanH region. The data presented in this study indicate that the polymorphism of Tn1546 can be exploited in tracking transmission routes, monitoring the dispersion of VanA elements and investigation of the evolution of VRE strains.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Tiruvoor G. Nagaraja / Copper, as copper sulfate, is often supplemented at elevated concentrations in swine diets, particularly in piglets, to promote growth. Growth promotional effects of copper are believed to be similar to that of antibiotics in that gut microbial flora is altered to reduce loss of nutrients and suppress pathogens. Bacteria exposed to copper may acquire resistance, and in Enterococcus faecium and E. faecalis, resistance is conferred by a plasmid-borne transferable copper resistance (tcrB) gene. The plasmid also carries macrolide [erm(B)] and glycopeptide (vanA) antibiotics resistance genes. The objectives of the research were to 1) determine the prevalence of tcrB gene in fecal enterococci of piglets in relation to normal (16.5 ppm) and elevated level (125 ppm) of copper supplementation, 2) determine the relationship of tcrB gene and susceptibilities to copper, erythromycin, and vancomycin, and 3) determine the transferability of tcrB gene in enterococci by conjugation. Weaned piglets, housed in pens, fed normal (16.5 ppm; control) or elevated level of copper (125 ppm) were used. Fecal samples were collected weekly for isolation of enterococci. Isolates were speciated by multiplex PCR and sodA gene sequence analysis. The prevalence of tcrB-positive enterococcal isolates was higher (P < 0.05) in the copper supplemented group than the control group. The prevalence of tcrB was affected by sampling days (P < 0.05) with a significant treatment and sampling time interaction (P < 0.05). The tcrB positive isolates were either E. faecium or E. faecalis, and majority of isolates was E. faecium. The mean MIC of copper for tcrB-positive isolates (21.1 mM) was higher (P < 0.001) compared to tcrB-negative isolates (6.1 mM). All isolates were resistant to erythromycin, tetracyclines and susceptible to vancomycin. The transferability of the tcrB gene from tcrB-positive strains to tcrB-negative strains was demonstrated by conjugation. The potential link between tcrB and antibiotic resistance genes and the propensity of enterococci to transfer tcrB to other strains suggests the possibility that copper supplementation may exert selection pressure for antibiotic resistance. The positive association between copper supplementation and prevalence of tcrB gene has important implications for antimicrobial resistance and food safety, which warrants further investigation.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / H. Morgan Scott / Copper, as copper sulfate, is often used as an alternative to in-feed antibiotics for growth promotion in both swine and cattle diets. Gut bacteria exposed to copper can acquire resistance, which among enterococci is conferred by a plasmid-borne transferable copper resistance gene (tcrB). The plasmid also carries tetracycline [tet(M)] and macrolide [erm(B)] resistance genes. Because of the genetic link between acquired copper (tcrB) and antibiotic resistance in Enterococcus spp., we hypothesized that copper supplementation may exert selection pressure for enterococci to become resistant to macrolides and tetracyclines, and possibly to other antibiotics. We conducted studies in cattle and swine to investigate the relationship between copper supplementation and the fecal prevalence of tcrB-positive enterococci, as well as its potential co-selection for macrolide and tetracycline resistance. The prevalence was higher in animals fed diets supplemented with elevated level of copper compared to normal level (P < 0.05). The tcrB-positive isolates belonged to either E. faecium or E. faecalis; the majority was E. faecium. All tcrB-positive isolates also contained both erm(B) and tet(M) genes; however, none of them harbored the vanA gene. Median minimum inhibitory concentrations (MIC) of copper for tcrB-positive and tcrB-negative enterococci were 22 mM and 4 mM, respectively (P < 0.0001). The overall prevalence of erm(B) and tet(M) genes among enterococcal isolates of cattle were 46.8 % and 57.5%, respectively; in contrast,100% of the swine isolates were positive for both erm(B) and tet(M) genes. The transferability of the tcrB gene was demonstrated by filter mating assay. Multi-locus variable number tandem repeat analysis revealed a genetically diverse population of enterococci. The finding of a strong association between the copper resistance gene and other antibiotic (tetracycline and tylosin) resistance determinants is significant because enterococci are potential pathogens and have the propensity to transfer resistance genes to other bacteria in the gut. The occurrence of vancomycin resistant enterococci in swine in the US is very rare. Strains of E. faecium positive for vanA, that confers resistance to vancomycin, were isolated and characterized from swine feces. The swine strains belonged to clonal complex 17, a well-adapted hospital clone throughout the world.

For production of double moulded chesses are used lactic acid bacteria, which can be present in a form of non-starter lactic acid bacteria or as starter or adjunct culture. Genera Lactobacillus spp. and Enterococcus spp. are prevalent microorganisms present in these cultures. Of course, these microorganisms are for us interesting because of their possibility of coagulation, proteolytic possibility, probiotic function and antibiotic resistance, but especially because of their decarboxylation abilities. Bacteria contain decarboxylation enzymes, which are able to decarboxylized free amino acid, which rising at proteolysis during process of manufacturing and cheese ripening. Biogenic amines are the result of proteolytic activity. Biogenic amines occur practically in all foodstuffs as a common product of metabolic processes. BA are mainly presented in fermented food (cheeses), where rice just microbial action. Typical representatives of biogenic amines, which occurs in double moulded cheeses (Sedlčanský Vltavín, Bresse bleu) and in blue cheeses (Bleu des Causses, Bleu d'Auvergne) are cadaverine, putrescine, tyramine a 2 fenylethylamine and in much smaller amount histamine, spermidine and spermine too. On assessment concentration of BA is used high pressure liquid chromatography with reverse phase (RP HPLC) with utilizing simple direct derivatization with dansyl chloride and detection by UV VIS detector.

Στην παρούσα μελέτη η βιοτυπία και η ανάλυση του DNA με ηλεκτρο-φόρηση σε παλλόμενο ηλεκτρικό πεδίο (PFGE) εφαρμόσθηκαν σε ένα σύνο-λο εντε-ρο-κόκ-κων που απομονώθηκαν από νερά αναψυχής και πόσιμα νερά, προκει-μένου να προσδιορισθεί πιθανή γενετική συγγένεια. Τα 200 στελέχη εντε-ρο--κόκκου απομονώθηκαν από 246 δείγματα νερών αναψυχής και 903 δείγματα πόσιμου νερού, από θάλασσα, νερό δικτύου, ποταμούς και πηγές της Ν.Δ. Ελλάδας. Η ταυτοποίηση σε επίπεδο είδους έγινε με το σύστημα API 20strep. Ο έλεγχος της ευαισθησίας στα αντιβιοτικά έγινε με προσδιορισμό της ελάχιστης ανασταλτικής πυκνότητας (MIC) με την μέθοδο ταινιών E-test. Η ηλεκτροφό-ρηση σε παλλόμενο ηλεκτρικό πεδίο εφαρμόσθηκε για τις γονοτυπικές δοκιμασίες. Ο χαρακτηρισμός σε επίπεδο είδους ταξινόμησε τους εντερόκοκκους σε 22 βιότυπους. Στην πλειοψηφία τους τα στελέχη ήσαν E. faecium (142 ή 71%), ακολουθούσε ο E. faecalis (40 ή 20%), o E. durans (9 ή 4,5%), o E. gallinarum (5 ή 2,5%) και o E. avium (4 ή 2%). Μεταξύ των στελεχών του E. faecium υπήρ-χε σχέση των βιοτύπων και της πηγής του δείγματος. Αυτό δεν παρατηρή-θη-κε στα στελέχη του E. faecalis ούτε στα υπόλοιπα είδη. Κανένα στέλεχος δεν βρέθηκε να παράγει β-λακταμάση. Δεν παρατηρήθηκε καμία σχέση μεταξύ της αντοχής στα αντιβιοτικά και της προέλευσης των στελεχών. Κανένα είδος εντεροκόκκου δεν παρουσίασε αντοχή στα γλυκοπεπτίδια. Κλινικά στελέχη E. faecium περιελήφθησαν στα γονοτυπικά πειράματα ως μάρτυρες για τους ήδη ταυτοποιημένους κλώνους που ενδημούν στο Πανεπιστημιακό Νοσοκομείο της Πάτρας. Η ανάλυση των τύπων PFGE μεταξύ των 104 στελεχών αποκάλυψε την παρουσία 18 κλώνων μεταξύ του E. faecium, 14 του E. faecalis, 2 του E. durans, 3 του E. gallinarum και ενός του E. avium. Αν και μεταξύ των εξετασθέ-ντων στελεχών παρατηρήθηκε γενετική ποικιλία, προσδιορίσθηκαν κοινοί κλώ-νοι μεταξύ διαφορετικών δειγμάτων νερών. Τα δενδρογράμματα αποκά-λυ-ψαν γενετική σχέση μεταξύ συγκεκριμέ-νων περιβαλλοντικών στελεχών του E. faecium και αυτών που ήσαν νοσοκομειακής προέλευσης. Για τη μελέτη της παρουσίας κλώνων μεταξύ εντεροκόκκων που απομονώνονται από το περιβάλλον πρέπει να εφαρμόζονται μέθοδοι ανάλυσης χρωμοσωμικού DNA. / In this study we have identified the enterococcal species isolated from different environmental sources and we have characterized their biotypes, antibiotic resistance patterns and PFGE types. Biotyping and DNA fingerprinting by pulsed-field gel electrophoresis was applied to a collection of enterococci recovered form recreational and drinking water, in order to identify possible genetic relationships. Clinical strains of hospital origin were compared to the environmental isolates. A total of 200 enterococci were isolated from 246 recreational water, and 900 drinking water. One hundred forty two isolates were characterized as Enterococcus faecium recovered from all sources, 40 E. faecalis, 9 E. durans, 5. E. gallinarum and E. avium. Biotypes, determined with API 20 strep, among E. faecium were correlated with certain environmental sources, while antibiotypes, determined with Etest, did not reveal any relationship with the sample origin. Even though genetic diversity was observed among the studied strains, common clonal types were also identified in different sources, suggesting a possible common origin of the enterococci. Cluster analysis revealed a genetic relationship between certain environmental E. faecium and clinical strains.

Introduction: Deep sternal wound infection is a rare but feared complication of median thoracotomies and is usually caused by microorganisms from the patient’s skin or mucous membranes, the external environment, or iatrogenic procedures. The most common involved pathogens are Staphylococcus aureus, Staphylococcus epidermidis and gram-negative bacteria. We aimed to evaluate the microbiological spectrum of deep sternal wound infections in our institution and to establish diagnostic and treatment algorithms. Methods: We retrospectively evaluated the patients with deep sternal wound infections at our institution between March 2018 and December 2021. The inclusion criteria were the presence of deep sternal wound infection and complete sternal osteomyelitis. Eighty-seven patients could be included in the study. All patients received a radical sternectomy, with complete microbiological and histopathological analysis. Results: In 20 patients (23%) the infection was caused by S. epidermidis, in 17 patients (19.54%) by S. aureus, in 3 patients (3.45%) by Enterococcus spp., in 14 patients (16.09%) by gram-negative bacteria, while in 14 patients (16.09%) no pathogen could be identified. In 19 patients (21,84%) the infection was polymicrobial. Two patients had a superimposed Candida spp. infection. Methicillin-resistant S. epidermidis was found in 25 cases (28,74%), while methicillin-resistant S. aureus was isolated in only three cases (3,45%). The average hospital stay for monomicrobial infections was 29.93 ± 13.69 days and for polymicrobial infections was 37.47 ± 19.18 (p = 0.03). Wound swabs and tissue biopsies were routinely harvested for microbiological examination. The increasing number of biopsies was associated with the isolation of a pathogen (4.24 ± 2.22 vs. 2.18 ± 1.6, p < 0,001). Likewise, the increasing number of wound swabs was also associated with the isolation of a pathogen (4.22 ± 3.34 vs. 2.40 ± 1.45, p = 0.011). The median duration of antibiotic treatment was 24.62 (4–90) days intravenous and 23.54 (4–70) days orally. The length of antibiotic treatment for monomicrobial infections was 22.68 ± 14.27 days intravenous and 44.75 ± 25.87 days in total and for polymicrobial infections was 31.65 ± 22.29 days intravenous (p = 0.05) and 61.29 ± 41.45 in total (p = 0.07). The antibiotic treatment duration in patients with methicillin-resistant Staphylococci as well as in patients who developed an infection relapse was not significantly longer. Conclusion: S. epidermidis and S. aureus remain the main pathogen in deep sternal wound infections. The number of wound swabs and tissue biopsies correlates with accurate pathogen isolation. With radical surgical treatment, the role of prolonged antibiotic treatment remains unclear and should be evaluated in future prospective randomized studies.

[ES] La amenaza para la prevención y tratamiento de las infecciones bacterianas provocada por la resistencia a los antibióticos es cada vez mayor. En este trabajo hemos estudiado distintos tipos de agua, a fin de obtener información integrada de la contaminación y los patrones locales de resistencia a antibióticos de bacterias indicadoras, con el objetivo de determinar el potencial de estos sistemas acuáticos para actuar como medios de dispersión de determinantes de resistencias a antibióticos en el ambiente, dentro de la perspectiva "One Health". Nuestro objetivo era obtener información acerca del nivel de contaminación por bacterias resistentes a antibióticos (ARB) y la presencia de genes determinantes de resistencias (ARG) en distintos tipos de aguas ambientales, evaluando el origen de esta resistencia, relacionando los patrones de resistencia presentes, valorando los posibles riesgos para la población y proponiendo medidas para combatir el problema. En las muestras de agua recogidas en diversas localizaciones realizamos recuentos bacterianos selectivos de bacterias consideradas indicadores de contaminación fecal, tales como los coliformes totales, Escherichia coli y Enterococcus spp. (enterococos), seleccionados dependiendo del origen de la muestra, con el fin de conocer su calidad higiénico-sanitaria. Empleando E. coli como microorganismo monitor de la resistencia en todos los tipos de agua, junto con Enterococcus spp. en el caso de aguas continentales y marinas, seleccionamos y aislamos un número significativo de cepas, valorando su concentración mínima inhibitoria (CMI) frente a antibióticos de uso frecuente para el tratamiento de infecciones en humanos. Por último, verificamos mediante PCR la presencia o ausencia de diferentes genes de resistencia, tanto en el medio acuático como en las cepas seleccionadas. Los resultados obtenidos reflejan que la calidad higiénico-sanitaria de las aguas procedentes de los efluentes de dos EDAR, aguas de mar y aguas continentales, están dentro de los estándares de calidad establecidos por la normativa española. No ocurre lo mismo con las aguas de riego y las aguas de una de las EDAR, las cuales estaban muy por encima de esos límites. Mediante recuento en medios selectivos adicionados con distintos antibióticos, se observó que un elevado porcentaje de las cepas provenientes de EDAR y aguas de riego eran resistentes a ampicilina, eritromicina, y sulfametoxazol, siendo la resistencia a la ampicilina la más prevalente. Estos resultados fueron confirmados por la CMI en los aislados de E. coli, los cuales mostraron un patrón global de resistencia independientemente del origen de la muestra, siendo la resistencia a ampicilina la más prevalente, seguida de tetraciclina y sulfametoxazol, con un porcentaje muy alto de cepas multirresistentes, especialmente en EDAR y aguas de riego. La mayoría de las cepas presentaron resistencia a los antibióticos betalactámicos, sulfonamidas y tetraciclinas, siendo los perfiles predominantes de multirresistencia similares en todos los tipos de agua. En el caso de Enterococcus spp., el número de cepas resistentes y multirresistentes fue escaso, siendo las resistencias a la eritromicina y tetraciclina las más prevalentes. La determinación por PCR de la presencia de diversos genes de resistencia confirmó que su dispersión en el medio acuático es muy elevada, encontrándose casi todos ellos en todas las muestras de agua, aunque en un menor porcentaje en el agua de mar. Se pudo determinar la existencia de un patrón genómico homogéneo de resistencias en las cepas de E. coli en esta área geográfica. La elevada prevalencia de determinantes de resistencia a los antibióticos encontrados en los distintos tipos de agua supone un riesgo elevado de transferencia horizontal de genes a otras bacterias, y contribuye de forma determinante a su llegada al ser humano. / [CA] L'amenaça per a la prevenció i el tractament de les infeccions bacterianes provocada per la resistència als antibiòtics és cada vegada més gran. En aquest treball hem estudiat diferents tipus d'aigua per obtenir informació integrada de la contaminació i els patrons locals de resistència a antibiòtics de bacteris indicadors, amb l'objectiu de determinar el potencial d'aquests sistemes aquàtics per actuar com a mitjans de dispersió de determinants de resistències a antibiòtics a l'ambient, dins de la perspectiva "One Health". El nostre objectiu era obtenir informació sobre el nivell de contaminació per bacteris resistents a antibiòtics (ARB) i la presència de gens determinants de resistències (ARG) en diferents tipus d'aigües ambientals, avaluant l'origen d'aquesta resistència, relacionant els patrons de resistència presents, valorant els possibles riscos per a la població i proposant mesures per combatre el problema. A les mostres d'aigua recollides en diverses localitzacions realitzem recomptes bacterians selectius de bacteris considerats indicadors de contaminació fecal, com ara els coliformes totals, Escherichia coli i Enterococcus spp. (enterococs), seleccionats depenent de l'origen de la mostra, per tal de conèixer-ne la qualitat higienicosanitària, i valorem el nivell de resistències d'aquests microorganismes davant de diferents antibiòtics d'ús freqüent, mitjançant recomptes en plaques amb medi selectiu a les quals s'havia afegit concentracions estàndard dels mateixos. Emprant E. coli com a microorganisme monitor de la resistència en tots els tipus d'aigua, juntament amb Enterococcus spp. en el cas d'aigües continentals i marines, seleccionem i aïllem un nombre significatiu de ceps, valorant-ne la concentració mínima inhibitòria (CMI) davant d'antibiòtics d'ús freqüent per al tractament d'infeccions en humans. Finalment, verifiquem mitjançant PCR la presència o absència de diferents gens de resistència, tant al medi aquàtic com als ceps seleccionats. Els resultats obtinguts reflecteixen que la qualitat higienicosanitària de les aigües procedents dels efluents de dues EDAR, aigües de mar i aigües continentals, estan dins dels estàndards de qualitat establerts. No passa el mateix amb les aigües de reg i les aigües d'una de les EDAR, que estaven molt per sobre d'aquests límits. Mitjançant recompte en mitjans selectius addicionats de diferents antibiòtics, es va observar que un elevat percentatge dels ceps provinents d'EDAR i aigües de reg eren resistents a ampicil·lina, eritromicina, i sulfametoxazol, sent la resistència a l'ampicil·lina la més prevalent. Aquests resultats van ser confirmats per la CMI als aïllats d' E. coli, els quals van mostrar un patró global de resistència independentment de l'origen de la mostra, sent la resistència a l'ampicil·lina la més prevalent, seguida de tetraciclina i sulfametoxazol, amb un percentatge molt alt de ceps multiresistents, especialment a EDAR i aigües de reg. La majoria de les ceps van presentar resistència als antibiòtics betalactàmics, sulfonamides i tetraciclines, sent els perfils predominants de multiresistència similars en tots els tipus d'aigua. En el cas d' Enterococcus spp., el nombre de ceps resistents i multiresistents va ser escàs, sent les resistències a l'eritromicina i tetraciclina les més prevalents. La determinació per PCR de la presència de diversos gens de resistència va confirmar que la seva dispersió en el medi aquàtic és molt elevada, i gairebé tots es troben en totes les mostres d'aigua, encara que en un menor percentatge a l'aigua de mar. Es va poder determinar l'existència d'un patró genòmic homogeni de resistències als ceps d' E. coli en aquesta àrea geogràfica. L'elevada prevalença de determinants de resistència als antibiòtics trobats en els diferents tipus d'aigua suposa un risc elevat de transferència horitzontal de gens a altres bacteris, i contribueix de manera determinant a l'arribada a l'ésser humà. / [EN] The threat to prevention and treatment of bacterial infections caused by antibiotic resistance is increasing. In this work we have studied different types of water, in order to obtain integrated information on pollution and local antibiotic resistance patterns of indicator bacteria, with the aim of determining the potential of these aquatic systems to act as means of dispersion of determinants of antibiotic resistance in the environment, within the "One Health" perspective. Our objective was to obtain information about the level of contamination by antibiotic-resistant bacteria (ARB) and the presence of resistance-determining genes (ARG) in different types of environmental waters, evaluating the origin of this resistance, relating the present resistance patterns, assessing the possible risks for the population and proposing measures to combat the problem. In water samples collected at various locations, we performed selective bacterial counts of bacteria considered indicators of fecal contamination, such as total coliforms, Escherichia coli and Enterococcus spp. (enterococci), selected depending on the origin of the sample, in order to know its hygienic-sanitary quality, and we assess the level of resistance of these microorganisms against different frequently used antibiotics, through counts on plates with selective medium to which standard concentrations of them had been added. Using E. coli as a monitoring microorganism of resistance in all types of water, together with Enterococcus spp. in the case of continental and marine waters, we selected and isolated a significant number of strains, assessing their minimum inhibitory concentration (MIC) against antibiotics frequently used for the treatment of infections in humans. Finally, we verified by PCR the presence or absence of different resistance genes, both in the aquatic environment and in the selected strains. The results obtained reflect that the hygienic-sanitary quality of the waters from the effluents of two WWTPs, sea waters and continental waters, are within the quality standards established by the Spanish. The same does not occur with irrigation water and water from one of the WWTPs, which were well above those limits. By counting in selective media added with different antibiotics, it was observed that a high percentage of the strains from WWTP and irrigation water were resistant to ampicillin, erythromycin, and sulfamethoxazole, with resistance to ampicillin being the most prevalent. These results were confirmed by the MIC in the E. coli isolates, which showed a global pattern of resistance regardless of the origin of the sample, with resistance to ampicillin being the most prevalent, followed by tetracycline and sulfamethoxazole, with a very high percentage. of multi-resistant strains, especially in WWTP and irrigation water. Most strains presented resistance to beta-lactam antibiotics, sulfonamides and tetracyclines, with the predominant multiresistance profiles being similar in all types of water. In the case of Enterococcus spp., the number of resistant and multiresistant strains was low, with resistance to erythromycin and tetracycline being the most prevalent. The determination by PCR of the presence of various resistance genes confirmed that their dispersion in the aquatic environment is very high, almost all of them were found in all water samples, although in a lower percentage in seawater. It was possible to determine the existence of a homogeneous genomic pattern of resistance in E. coli strains in this geographical area. The high prevalence of antibiotic resistance determinants found in different types of water represents a high risk of horizontal gene transfer to other bacteria, and contributes decisively to their arrival in humans. / Dasí Camaró, DM. (2024). Estudio del agua como fuente de entrada y dispersión de determinantes de resistencia a antibióticos al medio ambiente en el área metropolitana de Valencia y alrededores [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/210135