Microbial Complexity of Oral Cavity of Healthy Dogs Identified by Mass Spectrometry and Next-Generation Sequencing.

The high complexity of the oral microbiota of healthy dogs and the close exposure of humans to companion animals represent a risk of the transmission of potential zoonotic microorganisms to humans, especially through dog bites, including multidrug-resistant ones. Nonetheless, a limited number of comprehensive studies have focused on the diversity of the microorganisms that inhabit the oral cavities of healthy dogs, particularly based on modern molecular techniques. We investigated bacterial and fungal organisms in the oral cavities of 100 healthy dogs based on a combination of conventional and selective microbiological culture, mass spectrometry (MALDI-TOF MS), and next-generation sequencing. In addition, in vitro antimicrobial susceptibility patterns of isolates and mecA resistance gene were assessed. A total of 213 bacteria and 20 fungi were isolated. Staphylococcus pseudintermedius (40/100 = 40%), α-hemolytic Streptococcus (37/100 = 37%), and Pasteurella stomatis (22/100 = 22%) were the most prevalent bacteria diagnosed by microbiological culture and MALDI-TOF MS, whereas Aspergillus (10/100 = 10%) was the most common fungi identified. Based on next-generation sequencing of selected 20 sampled dogs, Porphyromonas (32.5%), Moraxella (16.3%), Fusobacterium (12.8%), Conchiformibius (9.5%), Bergeyella (5%), Campylobacter (3.8%), and Capnocytophaga (3.4%) genera were prevalent. A high multidrug resistance rate was observed in Staphylococcus pseudintermedius isolates, particularly to azithromycin (19/19 = 100%), penicillin (15/19 = 78.9%), and sulfamethoxazole/trimethoprim (15/19 = 78.9%). In addition, the mecA resistance gene was detected in 6.1% (3/49) of coagulase-positive staphylococci. Here, we highlight the microbial complexity of the oral mucosa of healthy dogs, including potential zoonotic microorganisms and multidrug-resistant bacteria, contributing with the investigation of the microbiota and antimicrobial resistance patterns of the microorganisms that inhabit the oral cavity of healthy dogs.

Characteristics of resistance and virulence factors in different species of coagulase-negative staphylococci isolated from milk of healthy sheep and animals with subclinical mastitis.

Coagulase-negative staphylococci (CNS) are among the main responsible agents for mastitis in sheep. Cure rates can be reduced due to several causes, such as those related to virulence factors presented by microorganisms. This study aims at characterizing the virulence and resistance factors to antimicrobial agents in different CNS species isolated from sheep milk. After collecting milk samples, the samples were analyzed and the CNS species were identified. After identification, the susceptibility-sensitivity profile was examined using the disk diffusion technique for 10 antimicrobial agents. The DNA was extracted to detect the presence of the mecA gene, biofilm (icaADBC, bap, and bhp) and toxin genes (sea, seb, sec, sed, tst, and luk-PV) by PCR. Samples carrying toxin genes had their expression assessed using the reverse-transcription PCR technique. The biofilm production was assessed using the adherence method on a polystyrene plate. One hundred twelve CNS samples were isolated, 53 (47.3%) from animals with subclinical mastitis and 59 (52.7%) from healthy animals. Drugs tested have shown to be efficient for most CNS samples. The largest resistance percentage of CNS was found for the penicillin (17.0%) and tetracycline (10.7%) and 4 samples carried the mecA gene. As for the biofilm genes, the icaADBC operon was found in 10 (8.9%) samples, the bap gene was found in 16 (14.3%), and the bhp gene was found in 3 (2.7%). In addition, 69 (61.6%) samples produced biofilm. The survey of toxin genes has shown that 70 (62.5%) samples showed some toxin-encoding gene. However, none of the samples has expressed any of the genes from those toxins studied.

Peritoneal dialysis-related peritonitis due to Staphylococcus aureus: a single-center experience over 15 years.

Peritonitis caused by Staphylococcus aureus is a serious complication of peritoneal dialysis (PD), which is associated with poor outcome and high PD failure rates. We reviewed the records of 62 S. aureus peritonitis episodes that occurred between 1996 and 2010 in the dialysis unit of a single university hospital and evaluated the host and bacterial factors influencing peritonitis outcome. Peritonitis incidence was calculated for three subsequent 5-year periods and compared using a Poisson regression model. The production of biofilm, enzymes, and toxins was evaluated. Oxacillin resistance was evaluated based on minimum inhibitory concentration and presence of the mecA gene. Logistic regression was used for the analysis of demographic, clinical, and microbiological factors influencing peritonitis outcome. Resolution and death rates were compared with 117 contemporary coagulase-negative staphylococcus (CoNS) episodes. The incidence of S. aureus peritonitis declined significantly over time from 0.13 in 1996-2000 to 0.04 episodes/patient/year in 2006-2010 (p = 0.03). The oxacillin resistance rate was 11.3%. Toxin and enzyme production was expressive, except for enterotoxin D. Biofilm production was positive in 88.7% of strains. The presence of the mecA gene was associated with a higher frequency of fever and abdominal pain. The logistic regression model showed that diabetes mellitus (p = 0.009) and ß-hemolysin production (p = 0.006) were independent predictors of non-resolution of infection. The probability of resolution was higher among patients aged 41 to 60 years than among those >60 years (p = 0.02). A trend to higher death rate was observed for S. aureus episodes (9.7%) compared to CoNS episodes (2.5%), (p = 0.08), whereas resolution rates were similar. Despite the decline in incidence, S. aureus peritonitis remains a serious complication of PD that is associated with a high death rate. The outcome of this infection is negatively influenced by host factors such as age and diabetes mellitus. In addition, ß-hemolysin production is predictive of non-resolution of infection, suggesting a pathogenic role of this factor in PD-related S. aureus peritonitis.

Peritonitis in recent years: clinical findings and predictors of treatment response of 170 episodes at a single Brazilian center.

PURPOSE: Peritonitis continues to be a major cause of dropout in peritoneal dialysis (PD) patients. Changes in the prevalence of peritonitis etiologies and an increase in the frequency of methicillin-resistant coagulase-negative staphylococci (CoNS) and Gram-negative species resistant to commonly used antibiotics have been reported. As a consequence, the current clinical presentation and patient outcome may differ from classical descriptions. The objectives of this study were to describe the clinical and microbiological characteristics of PD-related peritonitis episodes that occurred over a period of 6 years at a single Brazilian dialysis center and to identify predictors of outcome. METHODS: A total of 170 peritonitis episodes that occurred in 92 PD patients between January 2004 and December 2009 were reviewed. Multivariate analysis was used to identify demographic, clinical, and microbiological factors predicting endpoints (resolution and peritonitis-related death). Patients' characteristics and peritonitis incidence, etiology and outcomes were compared with a series of 232 episodes that occurred in the first 6 years (1995-2000) of the PD program at the same center. RESULTS: The overall peritonitis rate was 0.65 episodes/patient-year. Gram-positive cocci were identified in 79 (46.5%) episodes, whereas Gram-negative bacilli were isolated from 48 (28.2%). CoNS were the main Gram-positive species identified in 48 episodes. Of these, 56.3% were resistant to methicillin. Among Gram-negative species, amikacin resistance was observed in 60% of non-fermentative Gram-negative bacilli (NFGNB) and in only 3.6% of Enterobacteriaceae. The overall resolution rate was 44.1%. Oxacillin resistance and NFGNB etiology were strong predictors of non-resolution, whereas older age was the only predictor of death. Antibiotic protocols did not influence outcome. Comparison with the results obtained for the 1990-1995 period showed a lower peritonitis rate, a strong decline in the proportion of Staphylococcus aureus episodes, a significant increase in the frequency of oxacillin-resistant CoNS and amikacin-resistant NFGNB, and a significantly lower resolution and higher death rate. CONCLUSIONS: The current clinical characteristics and outcome suggest a greater severity of peritonitis episodes and higher risk of death, possibly due to bacterial resistance. Older age is a risk factor for death.

Oxacillin resistance and antimicrobial susceptibility profile of Staphylococcus saprophyticus and other staphylococci isolated from patients with urinary tract infection.

BACKGROUND: Staphylococcus saprophyticus is the second most frequent community-acquired causative agent of urinary tract infection (UTI). The objective of this study was to evaluate the susceptibility profile and resistance detection in Staphylococcus species. isolated from patients with UTI. MATERIALS AND METHODS: The isolates were investigated using the disk diffusion method, Vitek I system, E-test®, and detection of the mecA gene. RESULTS: Most isolates (76.2%) were resistant to oxacillin by the disk diffusion method, followed by those resistant to penicillin (72.2%). The oxacillin disk diffusion method, E-test, and Vitek I method showed higher sensitivity (94.4%) and lower specificity (28.9, 26.5, and 24.0%, respectively) than the cefoxitin disk diffusion test (sensitivity: 83.5%, specificity: 85.5%) for the detection of oxacillin resistance. CONCLUSIONS: The large number of oxacillin-resistant isolates indicates that the breakpoint value recommended by the Clinical and Laboratory Standards Institute may overestimate oxacillin resistance in S. saprophyticus. Thus, changes in these guidelines are necessary for the correct detection of this resistance.

Oxacillin resistance of Staphylococcus aureus isolated from the university hospital of Botucatu Medical School in Brazil.

BACKGROUND: Oxacillin is the main drug used for the treatment of Staphylococcus aureus infections. However, resistance of S. aureus to oxacillin has become a major problem over recent decades. The aim of this study was to determine oxacillin resistance in S. aureus isolates obtained from the University Hospital of the Botucatu Medical School, Universidade Estadual Paulista, Brazil. MATERIALS AND METHODS: A total of 102 isolates collected between 2002 and 2006 were analyzed by detection of the mecA gene, cefoxitin and oxacillin disk diffusion methods, screening test on Mueller-Hinton agar and E-test. RESULTS: Forty-six (45.1%) isolates were mecA positive. The oxacillin disk diffusion method showed 86.9% sensitivity and 91.1% specificity. The screening method and cefoxitin disk diffusion presented a similar sensitivity (91.3%) and the same specificity. The E-test showed 97.8% sensitivity and the same specificity as observed for the other methods. CONCLUSIONS: The E-test yielded the best results compared to the other methods.

The role of virulence factors in the outcome of staphylococcal peritonitis in CAPD patients.

BACKGROUND: Peritonitis continues to be the most frequent cause of peritoneal dialysis (PD) failure, with an important impact on patient mortality. Gram-positive cocci such as Staphylococcus epidermidis, other coagulase-negative staphylococci (CoNS), and Staphylococcus aureus are the most frequent etiological agents of PD-associated peritonitis worldwide. The objective of the present study was to compare peritonitis caused by S. aureus and CoNS and to evaluate the factors influencing outcome. METHODS: Records of 86 new episodes of staphylococcal peritonitis that occurred between 1996 and 2000 in the Dialysis unit of a single university hospital were studied (35 due to S. aureus, 24 to S. epidermidis and 27 to other CoNS). The production of slime, lipase, lecithinase, nuclease (DNAse), thermonuclease (TNAse), alpha- and beta-hemolysin, enterotoxins (SEA, SEB, SEC, SED) and toxic shock syndrome toxin-1 (TSST-1) was studied in S. aureus and CoNS. Antimicrobial susceptibility was evaluated based on the minimal inhibitory concentration determined by the E-test. Outcome predictors were evaluated by two logistic regression models. RESULTS: The oxacillin susceptibility rate was 85.7% for S. aureus, 41.6% for S. epidermidis, and 51.8% for other CoNS (p = 0.001). Production of toxins and enzymes, except for enterotoxin A and alpha-hemolysin, was associated with S. aureus episodes (p < 0.001), whereas slime production was positive in 23.5% of CoNS and 8.6% of S. aureus strains (p = 0.0047). The first model did not include enzymes and toxins due to their association with S. aureus. The odds of resolution were 9.5 times higher for S. epidermidis than for S. aureus (p = 0.02) episodes, and were similar for S. epidermidis and other CoNS (p = 0.8). The resolution odds were 68 times higher for non-slime producers (p = 0.001) and were not influenced by oxacillin resistance among vancomycin-treated cases (p = 0.89). In the second model, the resolution rate was similar for S. aureus and S. epidermidis (p = 0.70), and slime (p = 0.001) and alpha-hemolysin (p = 0.04) production were independent predictors of non-resolution. CONCLUSION: Bacterial species and virulence factors rather than antibiotic resistance influence the outcome of staphylococcal peritonitis.

Comparison between qualitative and semiquantitative catheter-tip cultures: laboratory diagnosis of catheter-related infection in newborns / Comparação entre culturas qualitativa e semiquantitativa de ponta de cateter: diagnóstico laboratorial de infecção relacionada a cateter em recém-nascidos

This prospective study evaluated semiquantitative and qualitative catheter-culture methods for diagnosis of catheter-related infection (CRI) in newborns. Catheter tips from newborns admitted to the Neonatal Unit of the University Hospital of the Botucatu Medical School, UNESP were included in the study. Catheter cultures were performed with both semiquantitative and qualitative techniques. For CRI diagnosis, microorganisms isolated from catheter cultures and from peripheral blood cultures were identified and submitted to agent susceptibility test. The gold standard was the certain CRI diagnosis when same microorganism (specie and profile of susceptibility to agents) was isolated from both catheter tips and peripheral blood culture. A total of 85 catheters from 63 newborns were included in the study. The semiquantitative culture method, despite presenting lower sensitivity (90 percent), showed higher specificity (71 percent) when compared to 100 percent of sensitivity and 60 percent of specificity in the qualitative method. The identification of the microorganisms obtained from the catheter cultures showed a prevalence of coagulase-negative staphylococci(CNS) species. The specie Staphylococcus epidermidis (77.5 percent) was the prevalent in the catheters with positive semiquantitative cultures. Among 11 episodes with CRI diagnosis, 8 (72.7 percent) were associated with CNS species, of which 6 were S. epidermidis. Two episodes of CRI by S. aureus and one by Candida parapsilosis were also detected. The semiquantitative catheter-culture method showed advantages for CRI diagnosis in newborns when compared to the conservative qualitative method.

Este estudo prospectivo avaliou os métodos semiquantitativo e qualitativo de cultura de cateter para o diagnóstico de infecção relacionada a cateter (IRC) em recém-nascidos (RN). Foram incluídas pontas de cateteres provenientes de recém-nascidos internados na Unidade Neonatal do Hospital das Clínicas da Faculdade de Medicina de Botucatu, UNESP. Foram utilizadas as técnicas semiquantitativa e qualitativa de cultura de cateter. Para o diagnóstico de IRC, os microrganismos isolados das culturas de cateteres e de hemoculturas periféricas foram identificados e submetidos ao teste de sensibilidade a antimicrobianos. O padrão ouro correspondeu ao diagnóstico de certeza de IRC, com o isolamento do mesmo microrganismo (espécie e perfil de sensibilidade a antimicrobianos) isolado em hemocultura periférica. Foram estudados 85 cateteres provenientes de 63 RN. A cultura semiquantitativa, embora tenha apresentado menor sensibilidade (90 por cento), apresentou uma maior especificidade (71 por cento) em comparação à sensibilidade de 100 por cento e especificidade de 60 por cento encontradas na cultura qualitativa. Através da identificação dos microrganismos obtidos nas culturas de cateteres, observou-se uma predominância de espécies de Estafilococos coagulase-negativa (ECN). A espécie Staphylococcus epidermidis foi a prevalente (77,5 por cento) nos cateteres com culturas semiquantitativas positivas. Dos 11 episódios de IRC diagnosticados, 8 (72,7 por cento) foram associados a espécies de ECN, dos quais 6 eram da espécie S. epidermidis. Também foram detectados dois casos de IRC por S. aureus e um caso por Candida parapsilosis. O método de cultura semiquantitativo cateter apresentou vantagens para o diagnóstico de IRC em RN quando comparado com o método qualitativo tradicional.

Methicillin resistance in Staphylococcus aureus and coagulase-negative staphylococci: epidemiological and molecular aspects.

Infections caused by the genus Staphylococcus are of great importance for human health. Staphylococcus species are divided into coagulase-positive staphylococci, represented by S. aureus, a pathogen that can cause infections of the skin and other organs in immunocompetent patients, and coagulase-negative staphylococci (CNS) which comprise different species normally involved in infectious processes in immunocompromised patients or patients using catheters. Oxacillin has been one of the main drugs used for the treatment of staphylococcal infections; however, a large number of S. aureus and CNS isolates of nosocomial origin are resistant to this drug. Methicillin resistance is encoded by the mecA gene which is inserted in the SCC mec cassette. This cassette is a mobile genetic element consisting of five different types and several subtypes. Oxacillin-resistant strains are detected by phenotypic and genotypic methods. Epidemiologically, methicillin-resistant S. aureus strains can be divided into five large pandemic clones, called Brazilian, Hungarian, Iberian, New York/Japan and Pediatric. The objective of the present review was to discuss aspects of resistance, epidemiology, genetics and detection of oxacillin resistance in Staphylococcus spp., since these microorganisms are increasingly more frequent in Brazil.

Detection of enterotoxin and toxic shock syndrome toxin 1 genes in Staphylococcus, with emphasis on coagulase-negative staphylococci.

The detection of staphylococcal enterotoxins is decisive for the confirmation of an outbreak and for the determination of the enterotoxigenicity of strains. Since the recognition of their antigenicity, a large number of serological methods for the detection of enterotoxins in food and culture media have been proposed. Since immunological methods require detectable amounts of toxin, molecular biology techniques represent important tools in the microbiology laboratory. In the present study, polymerase chain reaction (PCR) was used to identify genes responsible for the production of enterotoxins and toxic shock syndrome toxin 1 (TSST-1) in S. aureus and coagulase-negative staphylococci (CNS) isolated from patients and the results were compared with those obtained by the reverse passive latex agglutination (RPLA) assay. PCR detection of toxin genes revealed a higher percentage of toxigenic S. aureus strains (46.7%) than the RPLA method (38.3%). Analysis of the toxigenic profile of CNS strains showed that 26.7% of the isolates produced some type of toxin, and one or more toxin-specific genes were detected in 40% of the isolates. These results suggests the need for further studies in order to better characterize the pathogenic potential of CNS and indicate that attention should be paid to the toxigenic capacity of this group of microorganisms.

Comparison of methods for the identification of coagulase-negative staphylococci

Coagulase-negative staphylococci (CNS) species identification is still difficult for most clinical laboratories. The scheme proposed by Kloos and Schleifer and modified by Bannerman is the reference method used for the identification of staphylococcal species and subspecies; however, this method is relatively laborious for routine use since it requires the utilization of a large number of biochemical tests. The objective of the present study was to compare four methods, i.e., the reference method, the API Staph system (bioMérieux) and two methods modified from the reference method in our laboratory (simplified method and disk method), in the identification of 100 CNS strains. Compared to the reference method, the simplified method and disk method correctly identified 100 and 99 percent of the CNS species, respectively, while this rate was 84 percent for the API Staph system. Inaccurate identification by the API Staph method was observed for Staphylococcus epidermidis (2.2 percent), S. hominis (25 percent), S. haemolyticus (37.5 percent), and S. warneri (47.1 percent). The simplified method using the simple identification scheme proposed in the present study was found to be efficient for all strains tested, with 100 percent sensitivity and specificity and proved to be available alternative for the identification of staphylococci, offering, higher reliability and lower cost than the currently available commercial systems. This method would be very useful in clinical microbiology laboratory, especially in places with limited resources.

Comparison of methods for the identification of coagulase-negative staphylococci.

Coagulase-negative staphylococci (CNS) species identification is still difficult for most clinical laboratories. The scheme proposed by Kloos and Schleifer and modified by Bannerman is the reference method used for the identification of staphylococcal species and subspecies; however, this method is relatively laborious for routine use since it requires the utilization of a large number of biochemical tests. The objective of the present study was to compare four methods, i.e., the reference method, the API Staph system (bioMérieux) and two methods modified from the reference method in our laboratory (simplified method and disk method), in the identification of 100 CNS strains. Compared to the reference method, the simplified method and disk method correctly identified 100 and 99% of the CNS species, respectively, while this rate was 84% for the API Staph system. Inaccurate identification by the API Staph method was observed for Staphylococcus epidermidis (2.2%), S. hominis (25%), S. haemolyticus (37.5%), and S. warneri (47.1%). The simplified method using the simple identification scheme proposed in the present study was found to be efficient for all strains tested, with 100% sensitivity and specificity and proved to be available alternative for the identification of staphylococci, offering, higher reliability and lower cost than the currently available commercial systems. This method would be very useful in clinical microbiology laboratory, especially in places with limited resources.

Significância clínica de estafilococos coagulase-negativa isolados de recém-nascidos / Clinical significance of coagulase-negative staphylococci isolated from neonates

Objetivo: avaliar a significância clínica de estafilococos coagulase-negativa (ECN) isolados de processos infecciosos em recém-nascidos da unidade neonatal do Hospital das Clínicas da Faculdade de Medicina de Botucatu.Método: as linhagens de ECN isoladas foram identificadas eclassificadas em significativas e contaminantes, com base em uma série de dados clínicos e laboratoriais obtidos dos prontuários dos pacientes internados na unidade neonatal. Foram pesquisados os dados referentes a fatores perinatais de risco para infecção, evolução clínica, alterações do hemograma e/ou positividade de proteína C-reativa e antibioticoterapia.Resultados: das 117 linhagens de ECN isoladas, 60 (51,3por cento)foram classificadas como significativas, e 57 (48,7por cento) como contaminantes. Das 54 crianças com infecção por ECN, 43 (79,6por cento) eram prematuras, e 27 (50,0por cento) tiveram peso ao nascimento < 1.500g. A maioria das crianças com infecção por ECN estava submetida a dois ou mais procedimentos invasivos (77,8por cento), incluindo o uso de cateter(88,9por cento), nutrição parenteral (64,8por cento) e ventilação mecânica (61,1por cento).O S.epidermidis foi a espécie mais freqUentemente isolada (77,8por cento), e mais associada com infecção (86,7por cento) do que com contaminação (68,4por centi). Outras espécies de ECN, incluindo duas linhagens de s.haemolyticus, três linhagens de s. lugdunensis, uma linhagem de S. simulans, uma de S. warneri e uma linhagem de S. xylosus também foram isoladas de crianças com evidência clínica de pneumonia, nterocolite necrosante e sepse.Conclusão: a maioria dos recém-nascidos com infecção por ECN apresentou...

[Clinical significance of coagulase-negative staphylococci isolated from neonates]. / Significância clínica de estafilococos coagulase-negativa isolados de recém-nascidos.

OBJECTIVE: To evaluate the clinical significance of coagulase-negative staphylococci (CNS) isolated from newborns' infections at Neonatal Unit of Hospital das Clínicas da Faculdade de Medicina de Botucatu. METHODS: The CNS strains isolated were identified and classified as clinically significant and contaminant, based on a series of clinical and laboratory data obtained from patients who stayed in the Neonatal Unit. The following data were analyzed: risk factors for infections, clinical evolution, abnormal blood cell counts and/or C-reactive protein and antibiotic therapy. RESULTS: Among the 117 CNS strains isolated, 60 (51.3%) were classified as significant and 57 (48.7%) as contaminant. Among the 54 infants infected by CNS, 43 (79.6%) presented very low birthweight (<1,500 g). Most of the infants infected by CNS were submitted to two or more invasive procedures (77.8%), including use of catheter (88.9%), parenteral nutrition (64.8%) and mechanical ventilation (61.1%). Staphylococcus epidermidis was the most frequently isolated species (77.8%) and more often associated with infection (86.7%) than with contamination (68.4%). Other species of CNS, including two strains of S. haemolyticus, three strains of S. lugdunensis, one strain of S. simulans, one strain of S. warneri and one strain of S. xylosus were also isolated from infants with clinical evidence of pneumonia, necrotizing enterocolitis and sepsis. CONCLUSIONS: Most newborns infected by CNS presented important risk factors for infection onset, including birthweight <1,500 g, foreign body presence and previous use of antibiotics. The identification of CNS species constitutes a useful marker of infection, since S. epidermidis was the species more frequently associated with infection.