The Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) identification versus biochemical tests: a study with enterobacteria from a dairy cattle environment

Abstract Mastitis adversely affects milk production and in general cows do not regain their full production levels post recovery, leading to considerable economic losses. Moreover the percentage decrease in milk production depends on the specific pathogen that caused the infection and enterobacteria are responsible for this greater reduction. Phenotypic tests are among the currently available methods used worldwide to identify enterobacteria; however they tend to misdiagnose the species despite the multiple tests carried out. On the other hand The Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) technique has been attracting attention for its precise identification of several microorganisms at species level. In the current study, 183 enterobacteria were detected in milk (n = 47) and fecal samples (n = 94) from cows, and samples from water (n = 23) and milk lines (n = 19). All these samples were collected from a farm in Rio de Janeiro with the specific purpose of presenting the MALDI-TOF MS technique as an efficient methodology to identify Enterobacteriaceae from bovine environments. The MALDI-TOF MS technique results matched the biochemical test results in 92.9% (170/183) of the enterobacteria species and the gyrB sequencing confirmed 100% of the proteomic technique results. The amino acid decarboxylation test made the most misidentifications and Enterobacter spp. was the most misidentified genus (76.9%, 10/13). These results aim to clarify the current biochemical errors in enterobacteria identification, considering isolates from a bovine environment, and show the importance for more careful readings of phenotypic tests which are often used in veterinary microbiology laboratories.

ß-lactamase-producing Gram-negative bacteria in an intensive care unit in southern Brazil

ABSTRACT The present study evaluated the antimicrobial susceptibility profile, ß-lactamase production, and genetic diversity of Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter spp. using phenotypic identification, antimicrobial susceptibility testing, and ß-lactamase phenotypic detection. Isolates were obtained from patients in an intensive care unit in a hospital in southern Brazil. Bacterial genomic DNA was extracted, followed by the genotypic detection of carbapenemases and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). Fifty-six isolates (26 Klebsiella pneumoniae, five Escherichia coli, three Enterobacter aerogenes, nine P. aeruginosa, and 13 Acinetobacter spp.) were evaluated. The phenotypic extended spectrum ß-lactamase (ESBL) test was positive in 53.8% of the K. pneumoniae isolates, 100.0% of the E. coli isolates, and 100.0% of the E. aerogenes isolates. Phenotypic and genotypic testing of K. pneumoniae carbapenemase (KPC) was positive in 50.0% of the K. pneumoniae isolates. Phenotypic and genotypic testing showed that none of the P. aeruginosa or Acinetobacter spp. isolates were positive for metallo- ß-lactamase (MBL). The bla OXA gene was detected only in Acinetobacter spp. The lowest genetic diversity, determined by ERIC-PCR, was observed among the KPC-producing K. pneumoniae isolates and OXA-producing Acinetobacter spp. isolates, indicating the inadequate dissemination control of multidrug-resistant bacteria in this hospital environment.

Enterobacteriaceae ISOLATES FROM THE ORAL CAVITY OF WORKERS IN A BRAZILIAN ONCOLOGY HOSPITAL / Enterobacteriaceae isoladas da cavidade bucal de trabalhadores de hospital oncológico do Centro-Oeste brasileiro

The evaluation of workers as potential reservoirs and disseminators of pathogenic bacteria has been described as a strategy for the prevention and control of healthcare-associated infections (HAIs). The aim of this study was to evaluate the presence of Enterobacteriaceae in the oral cavity of workers at an oncology hospital in the Midwest region of Brazil, as well as to characterize the phenotypic profile of the isolates. Saliva samples of 294 workers from the hospital’s healthcare and support teams were collected. Microbiological procedures were performed according to standard techniques. Among the participants, 55 (18.7%) were colonized by Enterobacteriaceae in the oral cavity. A total of 64 bacteria were isolated, including potentially pathogenic species. The most prevalent species was Enterobacter gergoviae (17.2%). The highest rates of resistance were observed for β-lactams, and 48.4% of the isolates were considered multiresistant. Regarding the enterobacteria isolated, the production of ESBL and KPC was negative. Nevertheless, among the 43 isolates of the CESP group, 51.2% were considered AmpC β-lactamase producers by induction, and 48.8% were hyper-producing mutants. The significant prevalence of carriers of Enterobacteriaceae and the phenotypic profile of the isolates represents a concern, especially due to the multiresistance and production of AmpC β-lactamases.

A investigação de trabalhadores dos serviços de saúde como reservatório e disseminadores de bactérias patogênicas tem sido referida como estratégia de prevenção e controle das infecções relacionadas à assistência à saúde. Este estudo buscou avaliar a presença de Enterobacteriaceae na cavidade bucal de trabalhadores de hospital oncológico do Centro-Oeste brasileiro, bem como caracterizar o perfil fenotípico dos isolados. Foi coletada amostra de saliva de 294 trabalhadores pertencentes às equipes de saúde e de apoio. Procedimentos microbiológicos foram realizados segundo técnicas referendadas. Dentre os participantes, 55 (18,7%) estavam colonizados por Enterobacteriaceae na cavidade bucal. Foram isoladas 64 bactérias, incluindo espécies potencialmente patogênicas. A espécie mais prevalente foi Enterobacter gergoviae (17,2%). As maiores taxas de resistências foram observadas para os β-lactâmicos e 48,4% dos isolados foram considerados multirresistentes. Para as enterobactérias pesquisadas, a produção de ESBL e KPC foi negativa. Porém, dentre os 43 isolados do grupo CESP, 51,2% foram considerados produtores de β-lactamase AmpC por indução e 48,8% mutantes hiperprodutores. Considera-se a prevalência de portadores de Enterobacteriaceae significativa e o perfil fenotípico dos isolados preocupante, especialmente pela multirresistência e produção de β-lactamases AmpC.

Heavy metal tolerance (Cr, Ag and Hg) in bacteria isolated from sewage

Samples of sewage from a university hospital and a chemistry technical school were analysed for the percentage of bacterial tolerance to chromium (Cr), silver (Ag) and mercury (Hg). Additionally, we investigated the effect of these metals on pigmentation and on some enzymatic activities of the metal tolerant strains isolated, as well as antimicrobial resistance in some metal tolerant Enterobacteriaceae strains. Tolerance to Cr was observed mainly in Gram positive bacteria while in the case of Ag and Hg the tolerant bacteria were predominately Gram negative. Hg was the metal for which the percentage of tolerance was significantly higher, especially in samples from the hospital sewage (4.1%). Mercury also had the most discernible effect on color of the colonies. Considering the effect of metals on the respiratory enzymes, one strain of Ag-tolerantBacillus sp. and one of Hg-tolerant P. aeruginosa were unable to produce oxidase in the presence of Ag and Hg, respectively, while the expression of gelatinase was largely inhibited in various Gram negative strains (66% by Cr). Drug resistance in Hg-tolerant Enterobacteriaceae strains isolated from the university hospital sewage was greater than 80%, with prevalence of multiple resistance, while the Ag-tolerant strains from the same source showed about 34% of resistance, with the predominance of mono-resistance. Our results showed that, despite the ability of metal tolerant strains to survive and grow in the presence of these elements, the interactions with these metals may result in metabolic or phisiological changes in this group of bacteria.

Enterobacteriaceae: produção de compostos sulfurados voláteis / Enterobacteriaceae: production of volate sulfur compounds

A halitose pode ser causada pelo metabolismo de algumas bactérias proteolíticas Gram-negativas que residem na cavidade bucal. Alguns estudos verificaram a produção dos compostos sulfurados voláteis (CSV) por bactérias dos gêneros Porphyromonas, Prevotella, Bacteroides, Treponema, Actinobacillus e Fusobacterium. O objetivo deste trabalho foi avaliar in vitro a capacidade de produção de CSV por algumas espécies de bactérias da família Enterobacteriaceae. Foram estudadas 37 cepas de bacilos Gram-negativos isolados da cavidade bucal, sendo nove Enterobacter sakazakii, nove Klebsiella pneumoniae, nove Enterobacter cloacae, cinco Chryseomonasluteola e cinco Escherichia coli. De cada cepa, foram preparadas oito culturas em Caldo Infuso Cérebro-Coração (BHI), sendo três para o teste de medições com halímetro e cinco para o teste organoléptico. As medições com o halímetro foram realizadas diretamente nos tubos de ensaio e os testes organolépticos foram feitos por cinco participantes devidamente treinados, obedecendo escala com escores de O a 5. No teste de medição com o halímetro, ocorreu maior produção de CSV por cepas de E. coli, seguida por E. cloacae. Cepas de E. sakazakii, K. pneumoniae e C. luteola apresentaram baixa produção de CSV. No teste organoléptico, os escores mais altos foram atribuídos a E. coli. Concluiu-se que as espécies testadas produziram CSV, sendo E. coli a espécie com maior produção desses compostos em ambos os testes.

Halitosis is influenced by the action of proteolitic Gram-negative bacteria which live in the oral cavity. In some previous studies, the production of volatile sulfur compounds (VSC) was observed by members from the gemes: Porphyromonas, Prevotella, Bacteroides, Treponema, Actinobacillus and Fusobacterium. The objective of this study was to examine VSC production by some species of the Enterobacteriaceae family. 37 strains of Enterobacteriaceae were isolated from oral cavities; nine Enterobacter sakazakii, nine Klebsiella pneumoniae, nine Enterobacter cloacae, five Chryseomonas luteola and five Escherichia coli. Eight cultures were obtained from each strain and inoculated into Brain Heart Infusion (BHI) broth, resulting in a total of 296 cultures. Three cultures were tested by means of a Halimeter® and five by means of organoleptic test. The Halimeter® measurements were performed directly from the open test tubes and the organoleptic measurements were determined by five odor judges by smelling the contents of the test tubes and scoring them on a scale of O to 5. The Halimeter® test showed a higher production of VSC by E. coli strains, followed by E. cloacae. However E. sakazakii, K. pneumoniae and C. luteola presented low VSC production. E. coli, produced the highest scores with the organoleptic test. In conclusion, all tested species yielded VSC, overall the specie with the highest production of these compounds in both tests was E. coli.

Coliform MPN counts of municipal raw sewage and sewage treatment plant in relation to the water of Buckingham Canal at Kalpakkam (Tamil Nadu, India).

Most Probable Number (MPN) of Total Coliforms (TC) and Faecal Coliforms (FC), and the physicochemical variables - temperature, Dissolved Oxygen (D.O.), Biochemical Oxygen Demand (B.O.D.), Chemical Oxygen Demand (C.O.D.), nitrates, phosphates and chlorides of municipal raw sewage and that of aeration tank and secondary clarifier of the Sewage Treatment Plant (STP), in relation to water at the treated sewage out-fall point, down-stream and up-stream of the Buckingham Canal at Kalpakkam were analyzed. Total Coliform and Faecal Coliform MPN counts were higher, 170 and 70/100 mL respectively in the raw sewage. However, the counts of the former in the aeration tank though remained similar, that of FC decreased to 50/100 mL; both of the counts further decreased to 30 and 44/100 mL respectively, in the secondary clarifier and were 110 and 23/100 mL, respectively at the treated sewage out-fall point in the canal. Total coliforms MPN was more than 18 times less in the water at the up-stream than that of the treated sewage out-fall point in the canal. Interestingly MPN of the FC in the up-stream water was nil while it was 8/100 mL in the canal's down-stream point. It is concluded that the FC, B.O.D., C.O.D., nitrates, phosphates and chlorides decreased and the D.O. increased in the treated-sewage due to the treatment of raw sewage through the STP.

Multiple antimicrobial resistance in Enterobacteriaceae isolates from pristine freshwater

A freshwater enterobacterial population (N = 111) was studied for antimicrobial and mercury resistance patterns, and for its possible association with biotic and abiotic factors in that environment. Conventional biochemical tests identified Klebsiella sp, Morganella sp, Serratia sp, Escherichia sp, Enterobacter sp, Edwarsiella sp, Proteus sp, Citrobacter sp, Providencia sp, and Kluyvera sp. There was no correlation between antimicrobial resistance patterns of isolates and bacterial genera, but resistance patterns varied among water samples and between seasons. Resistance to multiple antimicrobials was common (61%). The percentage of bacteria resistant to at least one antimicrobial differed between the rainy (100%) and dry seasons (89%). Resistance to â-lactams and chloramphenicol was the most frequent and resistance to amikacin, gentamicin and kanamycin was less frequent. The main water variables examined (abiotic factors pH and temperature; biotic factor chlorophyll a concentration) did not influence antimicrobial resistance. Significant impact on freshwater enterobacteria, as evidenced by antimicrobial-multiple resistance and by the presence of blaTEM gene, may point to the fact that it has an important role in horizontal spread of resistance.

A Case of Pneumonia Caused by Ewingella americana in a Patient with Chronic Renal Failure

Though the pathogenic significance and the reservoir of Ewingella americana have not been clarified, this organism has caused several pathogenic infections, especially in immunocompromised patients. We report a pneumonia in a patient with chronic renal failure, who had chronic rejection of transplanted kidney. E. americana was identified to be the pathogen of pneumonia with clinical symptoms and signs and radiological examination. As soon as he was treated with ceftriaxone and isepamicin, clinical improvement was followed with no further growth of E. americana or other pathogenic isolates from sputum culture. This suggests to be the case of pneumonia caused by E. americana for the first time in the Korean literature.

Reductive and hydrolytic activities of enteric bacteria on food colours.

Reductive and hydrolytic activities on two food colours namely metanil yellow and indigo carmine by different strains of enteric bacteria were evaluated. Enteric bacteria possessed both reductive and hydrolytic capabilities which depended upon the incubation period and the type of strain. In certain bacterial strains, (Escherichia coli and Vibrio sp.), reductive activity was visible after 3 hr. Other enteric strains showed an increasing activity right from the beginning of incubation. Enterobacter cloacae appeared to be producing maximum reductive capability followed by Enterobacter aerogenes and E. coli. Even enteric bacterial strains demonstrated a good response in obtaining hydrolytic activity of indigo carmine during incubation upto 10 hr, establishing the fact that the gut microflora may cause health problem to consumers.

Ocorrência de Shigella em alimentos e dificuldades associadas com a sua detecçäo / Occurrence of Shigella in food difficulties associated with its detection

Foi feita uma avaliaçäo da importância de Shigella como patógeno de origem alimentar e das principais dificuldades associadas com os métodos de detecçäo disponíveis. Também foram revistas algumas alternativas para o aprimoramento dessa metodologia, particularmente em relaçäo à garantia da recuperaçäo de células de Shigella submetidas à injúria subletal durante o processamento

Gas liquid chromatography of amines produced by the Enterobacteriaceae

1. Gas liquid chromatography (GLC) was used to test the production of amines by 85 strains of Enterobacteriaceae. 2. The strains tested produced cadaverine, ß-phenylethylamine, putrescine, iso-amylamine, 2-methylbutylamine and iso-butylamine. 3. Although the overlap in amine production between obviouasly different genera sspecies limits the general applicability of this methology in clinical microbiology, the results obtained limits the general applicability of this methodology in clinical microbiology, the results obtained demonstrate the possibility of differentiating Proteus rettgeri from other Proteus species, Escherichia coli from Proteus species and Salmonella species from Shigella species on the basis of their amine composition. 4. In general, members of the Enterobacteriaceae family produce qualitatively similar amine profiles, thought differing quantitatively in the amounts of individual amines produced. GLC proves to be useful for separating amines and for provididng tentative peak identification

Concentraçöes inibitórias mínimas de ciprofloxacin (BAY-9867) para bactérias isoladas no Rio de Janeiro, RJ / Minimal inhibitory concentrations of ciprofloxacin (BAY-9867) for isolated bacteria isolated in Rio de Janeiro, RJ

Foi determinada a concentraçäo inibitória mínima in vitro do ciprofloxacin para cepas de enterobactérias, enterecocos, staphylococcus, Pseudomonas e bactérias anaeróbias estritas isoladas de diversos processos infecciosos de pacientes do Rio de Janeiro, RJ. A maioria das cepas de Escherichia coli e Klebsiella pneumoniae foi inibida com 0,25microng/ml do antimicrobiano, enquanto 1,0microng/ml foi necessário para inibir 90% das cepas de Proteus e Staphylococcus. A CIM90 para cepas de Pseudomonas aeruginosa e Streptococcus faecalis atingiu 2,0microng/ml; no entanto, para bactérias anaeróbias estritas (Bacteroides fragilis e Clostridium perfringens) elevou-se para 16 e 18micron/ml, respectivamente. A análise da curva de morte empregando amostra-padräo de Escherichia coli (ATCC-25922) revelou o efeito bactericida do ciprofloxacin

Favorece la Giardia lamblia la proliferación intestinal de bacterias? / Does giardia lamblia favor intestinal bacterial proliferation?

Con el propósito de estudiar si la G. lamblia se acompaña en el intestino de una gran población de bacterias productoras de indol, se investigó la concentración de sulfato de indoxil en la orina, antes y después de erradicar este parásito. Los resultados plantean, indirectamente, que la giardiasis se asocia a una proliferación de enterobacterias que sobrepasa en magnitud la que ordinariamente existe en el intestino

Flora residente intestinal: funciones fisiológicas y alteraciones / Resident intestinal flora: physiological functions and changes

Después del nacimiento el lumen intestinal es colonizado rápidamente, el canal vaginal materno y el medio ambiente proveen las primeras poblaciones microbianas, que ingresan por vía oral y proliferan en los nichos ecológicos que se originan por interacción entre la mucosa intestinal y dichas poblaciones. En esófago, estómago y los tramos altos del intestino, la flora microbiana es escasa con predominio de los aerobios, luego en yeyuno e íleon las poblaciones son mayores, con la presencia de anaerobios, los que luego en los segmentos distales constituyen la mayor parte de la flora comensal del intestino. El control de la flora residente en el período neonatal está fuertemente influenciado por la lactancia materna, luego de esta etapa la dieta pasa a jugar un rol secundaio y el control de la flora es ejercido fundamentalmente por factores no específicos tales como la acidez gástrica, el peristaltismo intestinal, la presencia de mucus y glicoproteínas y factores inmunológicos específicos, presencia de IgA secretoria y células inmunocompetentes (Placas de Peyer, linfocitos, células M, etc.). Como resultado de este conjunto de mecanismos de control el intestino es colonizado por la denominada flora "normal", que es a su vez uno de los medios más relevantes de control en la colonización por patógenos. El estudio de los roles fisiológicos de la flora ha demostrado que esta se asocia a cambios morfológicos y funcionales del intestino. El metabolismo bacteriano repercute en algunas actividades metabólicas del ser humano, deconjugando hormonas sexuales, activando fármacos o desdoblando metabolitos inactivos. En ocasiones la flora lleva a situaciones no deseadas tales como deconjugación de sales biliares y esteatorrea, activación de carcinógeno, sustracción de la vitamina B12, etc. Las alteraciones de la naturaleza y número de poblaciones asociadas a la flora normal pueden desencadenar trastornos tales como el spruetropical o el síndrome de intestino contaminado, característico de sobrepoblación de bacterias en segmentos altos del intestino, diarrea y cuadros de mala absorción