Intestinal colonization of newborns treated in intensive care units by multiple drug resistant microorganisms

The aim of the study was to examine the digestive tract colonisation of the newborns by multiple drug resistant bacteria during hospitalization. On the day of admission, after 5 days of hospitalization and at the day of discharge swabs from the anus of the 31 newborns hospitalized in OITiPN were taken and cultured on nutrient and selective media for staphylococci, enterococci, gram negative bacilli and fungi. Susceptibility to antibiotics of bacteria was determined, with giving attention to such resistance mechanisms as: methicillin resistant staphylococci (MRS), high level aminoglycoside resistant (HLAR) and vancomycin resistant enterococci (VRE) and the production of extended spectrum beta-lactamases (type ESBL) by gram negative bacilli. On the day of admission in 7 newborns methicillin resistant staphylococci (MRSCN) were grown in 24 no multiple drug resistant bacteria were found. Among those in 23 already after 5 days of hospitalization, colonization by multiple drug resistant strains was determined: coagulase-negative methicillin resistant staphylococci (MRSCN) were found in 16 children, strains of enterococci (HLAR) in 3 newborns and gram negative ESBL (+) bacilli also in 3 cases. On the day of discharge from hospital (after 13-141 days) in 23 out of 24 newborns enteric tract colonization by multiple drug resistant strains was assessed. In the enteric tract of 3 newborns hospitalized up to 2 weeks coagulase-negative methicillin resistant staphylococci (MRSCN) and/or HLAR enterococci were found; gram negative bacilli that produce ESBL appeared in newborns hospitalized for longer than 14 days. They were isolated in 12 out of 21 newborns. Forming of the enteric tract bacterial flora of the long hospitalized newborns depends on the time of hospitalization as well as on the used therapy.     

Poly-beta-hydroxybutyrate in staphylococci.

Staphylococci--chemoorganotrophic bacteria whose main habitats are human and animal organisms--can accumulate poly-beta-hydroxybutyrate (PHB) in their cells. The polymer is metabolized in endogenous turnovers preceding degradation of aminoacids, proteins and RNA. PHB depolymerase was not found in staphylococci but beta-hydroxybutyrate dehydrogenase was estimated, purified and characterized.     

Poly-β-hydroxybutyrate in staphylococci

Abstract Staphylococci—chemoorganotrophic bacteria whose main habitats are human and animal organisms—can accumulate poly-β-hydroxybutyrate (PHB) in their cells. The polymer is metabolized in endogenous turnovers preceding degradation of aminoacids, proteins and RNA. PHB depolymerase was not found in staphylococci but β-hydroxybutyrate dehydrogenase was estimated, purified and characterized.     

Antagonistic action of corynebacteria and bacilli of a skin ecotype on staphylococci

582 strain of corynebacteria and 235 cultures of bacilli isolated from the skin surface of mammary glands of 120 nursing women were studied for their antagonistic action on staphylococci. It is established that bacilli, especially Bacillus subtilis, B. firmus and B. alvei exert more pronounced inhibitory effect on staphylococci. Representatives of the family Corynebacteriaceae possess a weak antagonistic action both on coagulase-positive and on coagulase-negative species. The ability of coryneform bacteria to retain the colonization resistance is supposed to be due to other factors. Antagonistic properties of B. subtilis are shown expedient to be used for elimination of hospital strains of Staphylococcus aureus from the skin of mammary glands.     

Evaluation of media for monitoring fecal streptococci in seawater

The selectivity of KF streptococcus agar (KF) for monitoring fecal streptococci (FS) in seawater was examined in 234 samples of Mediterranean water and compared with the selectivity of M-Enterococcus agar (M-Ent) for 124 samples and with bile-esculin-azide agar (BEA) for 17 samples. KF was found to be unsuitable for marine water because Vibrio alginolyticus and other gram-negative bacilli indigenous to this environment grew well on it and produced red colonies identical to those of FS. In 26% of samples, some with high counts of red colonies on the membrane filters (MF), there were no streptococci, only gram-negative bacilli and staphylococci, and in an additional 23.1% the streptococci constituted less than 50% of the "typical" red colonies on the MF. V. alginolyticus also produced FS-like colonies on MF incubated on BEA but was not isolated from MF incubated on M-Ent. Although staphylococci grew and produced FS-like colonies on all three media, M-Ent was the most selective since no gram-negative bacilli were isolated from MF incubated on it.     

Evaluation of media for monitoring fecal streptococci in seawater.

The selectivity of KF streptococcus agar (KF) for monitoring fecal streptococci (FS) in seawater was examined in 234 samples of Mediterranean water and compared with the selectivity of M-Enterococcus agar (M-Ent) for 124 samples and with bile-esculin-azide agar (BEA) for 17 samples. KF was found to be unsuitable for marine water because Vibrio alginolyticus and other gram-negative bacilli indigenous to this environment grew well on it and produced red colonies identical to those of FS. In 26% of samples, some with high counts of red colonies on the membrane filters (MF), there were no streptococci, only gram-negative bacilli and staphylococci, and in an additional 23.1% the streptococci constituted less than 50% of the "typical" red colonies on the MF. V. alginolyticus also produced FS-like colonies on MF incubated on BEA but was not isolated from MF incubated on M-Ent. Although staphylococci grew and produced FS-like colonies on all three media, M-Ent was the most selective since no gram-negative bacilli were isolated from MF incubated on it.     

Closely related plasmids from Staphylococcus aureus and soil bacilli

Antibiotic resistance plasmids from staphylococci and soil bacilli have been isolated and compared. A tetracycline resistance (Tc^r) plasmid, indistinguishable from pT181, which is typical of Tc^r plasmids that are widely dispersed among human clinical isolates of S. aureus, has been found also in bovine mastitis isolates. This plasmid, however, shows no detectable homology to a family of related Tc^r plasmids, typified by pBC16, that is widely dispersed among aerobic spore-forming bacilli. However, and rather unexpectedly, pBC16 is highly homologous to and incompatible with pUB110, an S. aureus plasmid specifying kanamycin resistance. The two plasmids are homologous except for the region occupied by their resistance determinants, which has the appearance of a heterologous substitution. These results suggest the occurrence of natural plasmid transfer between staphylococci and soil bacilli.     

Sensitivity to antibiotics of bacterial strains isolated from clinical specimens during the years 1985-1986

Sensitivity of 312 strains of staphylococci, 386 strains of streptococci and 1193 strains of aerobic gram-negative bacilli to the selected antibiotics was tested. These strains were isolated from the clinical material at the Clinical Hospital No. 1 in Warsaw within 1985-1986. Staphylococci were sensitive to pristinamycin, cefazolin, fusidic acid, oxacillin, and clindamycin. In 1986, a decrease in the number of strains sensitive to these antibiotics, except cefazolin, was seen. In case of streptococci the most active proved chloramphenicol and gentamicin but a significant decrease in the percentage of sensitive strains was also noted in 1986. The highest number of gram-negative bacilli was sensitive to amikacin, colistin, nalidixic acid, pipemidic acid, and gentamicin. In 1986, a decrease in the percentage of sensitive strains was noted. Amikacin and colistin were the most active against Pseudomonas spp. while amikacin and nalidixic and pipemidic acids--against Proteus spp. Comparison of the results with those obtained in 1981-1984 has shown that the sensitivity of staphylococci changed the most significantly and this change was unfavourable. Gentamicin and amikacin remained the most active against gram-negative bacilli while amikacin and colimycin against Pseudomonas spp. In case of anaerobes the majority of strains was sensitive to chloramphenicol, tetracycline and clindamycin. Metronidazole was active against high percentage of Clostridium spp. and all gram-negative bacilli while the percentage of gram-positive bacilli and cocci was sensitive to metronidazole.     

pSRD191, a new member of RepL replicating plasmid family from Selenomonas ruminantium

A numerous plasmid population was detected in strain 19 of Selenomonas ruminantium. The population was found to consist of six plasmids in size ranging from 1.4 to more than 20kb. The smallest 1.4kb cryptic plasmid pSRD191 was further characterized. Sequence analysis identified a single ORF encoding the 177-residue putative replication protein (Rep191) which shared significant homology with RepL family of replication protein from Firmicutes (staphylococci and bacilli). PCR analysis and Southern hybridisation showed that pSRD191 related plasmids are frequently encountered in rumen selenomonads.     

A survey of microorganisms for thermonuclease production

A total of 1204 cultures comprising 16 genera were surveyed for production of thermonuclease (TNase) in milk. Cultures other than Staphylococcus capable of TNase production were restricted to two genera, Streptococcus and Bacillus. Nineteen percent of 338 group D streptococci comprising four species (85% of which were Streptococcus faecalis) and 17% of 60 streptococci belonging to other groups produced TNase. Nine percent of 130 Bacillus cultures comprising six species produced the enzyme. On the other hand, 99% of coagulase-positive staphylococci produced TNase and only 18% of the coagulase-negative staphylococci produced the enzyme. The amount of TNase produced by streptococci and bacilli was significantly lower than that produced by coagulase-positive staphylococci. The pH profile of the streptococci and Bacillus TNases was similar to that of the staphylococcal TNase; each enzyme exhibited a minor peak at pH 7.0 and a broad major peak ranging from pH 8.5 to 10. The nuclease produced by coagulase-positive Staphylococcus was more heat stable than the nucleases produced by Streptococcus and Bacillus; there was little loss in activity of the staphylococcal enzyme after 60 min at 100 degrees C, whereas 50% of the activity of the streptococcal and Bacillus nucleases was destroyed in 40-60 min and 60-80 min, respectively.     

Bactericidal activity of the membrane fraction isolated from phagocytes of mice and its stimulation by melittin

The membrane fraction prepared from mouse peritoneal exudate cells was incubated with mycobacteria, staphylococci, or E. coli in acetate buffer of pH 5.6 to follow the fate of viable bacilli. The membrane fraction exhibited bactericidal effect on mycobacteria and staphylococci, but not on E. coli. The activity to kill mycobacteria, as well as the endogenous phospholipase A2 activity, of the membrane fraction was markedly enhanced by melittin, a basic peptide from bee venom, and inhibited by indomethacin and EDTA. The role of the enzyme activity in the bactericidal activity was discussed.     

Effect of pH on the Proportions of Polar Lipids, in Chemostat Cultures of Bacillus subtilis

Significant changes in the relative proportions of the individual polar lipids of two strains of Bacillus subtilis were observed when the pH of their chemostat cultures was varied. In phosphate- and magnesium-limited cultures of B. subtilis var. niger NCIB 8058. lysylphosphatidylglycerol was present in higher proportions at low pH (5.1) than at neutral pH. With magnesium-limited cultures of this strain harvested at pH 8.0, lysylphosphatidylglycerol and phosphatidylethanolamine were not detected. Phosphate-limited cultures of B. subtilis NCIB 3610 contained no phosphatidylethanolamine or lysylphosphatidylglycerol at neutral pH, but at low pH (5.1) both these lipids were present in substantial proportions. The proportions of phosphatidylglycerol in actively dividing cells of chemostat cultures of bacilli were always greater than those of lysylphosphatidylglycerol. The reverse is commonly found in batch cultures of bacilli and staphylococci harvested at low pH. Changes in the proportions of the other polar lipids present in these bacilli (diphosphatidylglycerol and diglucosyl diacylglycerol) with pH were also noted. Certain cultures of both strains of B. subtilis contained small proportions of a peptidolipid.     

Antagonistic action of Pseudomonas aeruginosa against Staphylococci, coliform bacilli and various types of Proteus

Antagonistic activity of 2 fresh isolates and 3 collection strains of Pseudomonas aeruginosa against 177 microbial strains was determined with the method of late antagonism. Among the microbial strains there were 56 staphylococcal strains isolated from patents and carriers. 38 nontypable colon bacilli isolated from healthy persons, 59 enteropathogenic colon bacilli of various serogroups, 12 strains of Proteus and 12 colon bacilli, carriers of multiple drug resistance factors (R factors). All the cultures were sensitive to the antagonistic action of 5 or at least 3 strains of Pseudomonas used in the study. The most active antagonists were the fresh isolates of Pseudomonas as compared to the collection strains. Among the staphylococci S. aureus proved to be the most resistant to the antagonistic action of Pseudomonas as compared to S. epidermidis, the same as the strains isolated from carriers as compared to the strains isolated from patients. As for the enteric bacilli the most resistant were the strains of Proteus. Acquiring of transmissive R factors by the colon bacilli markedly increased their sensitivity to the antagonistic action of Pseudomonas.     

Bacterial colonization of patients treated with chronic hemodialysis]

The study was aimed at investigating the predispositions of the patients chronically treated with hemodialysis to nose and skin colonization with potentially pathogenic bacteria. The study involved 41 patients chronically hemodialysed and patients treated at the Department of Renal diseases or out-patient clinic (30 individuals). Smears from the nose and throat were taken from all patients and used for bacteriologic tests. In case of hemodialysed patients material for bacteriologic tests was additionally taken from the skin at the site of arterio-venous fistula and groin. It was found, that patients chronically treated with hemodialyses are more frequently colonized with enteric bacilli and coagulase-positive staphylococci that both hospitalized patients and those treated in out-patient clinic. No relationship between the presence of coagulase-positive staphylococci in the nose and throat and on the skin was seen despite such suggestions of other authors.     

富养罗尔斯通氏菌菌株PHB"泄漏"机理的初探

建立了一种分离纯化聚羟基丁酸(Polyhydroxybutyrate,PHB)颗粒的改良方法。采用这种方法从Ralstonia eutropha菌株H16(野生型)、SK1489(Tn5诱变的PHB泄漏菌株)、JMP222(野生的PHB泄漏菌株)分离了PHB颗粒。进一步比较研究了不同菌株的PHB解聚酶和3-羟基丁酸脱氢酶的活性。研究结果表明,菌株SK1489的PHB解聚酶活性(48h培养后达1．82U／mg)明显高于野生型菌株H16(48h培养后达0．37U／mg),菌株JMP222的3-羟基丁酸脱氢酶活性(培养96h后达165．9U／mg)比菌株H16培养(96h后达64．0U／mg)高许多。这些结果显示,不同菌株PHB的泄漏有不同的原因,突变株SK1489导致PHB泄漏的原因是解聚酶活性高,而野生型JMP222PHB泄漏的原因主要是3-羟基丁酸脱氢酶活性高。     

Drug sensitivity of bacterial strains isolated from clinical specimens during the years 1987-1988

Sensitivity to selected chemotherapeutics of various bacterial strains was analysed. Bacteria were isolated from the different material collected from patients within 1987-1988, and included: 690 strains of staphylococci, 465 strains of streptococci, 1224 strains of aerobic gram-negative bacilli, and 163 strains of anaerobic micro-organisms. Out of isolated staphylococci, the highest percentage was sensitive to vancomycin, pristinamycin, and fusidic acid. Vancomycin proved the most effective against streptococci followed by chloramphenicol and netilmicin . However, streptococci were highly sensitive to vancomycin within two years whereas their sensitivity to chloramphenicol and netilmicin .     

Properties of a novel intracellular poly(3-hydroxybutyrate) depolymerase with high specific activity (PhaZd) in Wautersia eutropha H16

A novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase (PhaZd) of Wautersia eutropha (formerly Ralstonia eutropha) H16 which shows similarity with the catalytic domain of the extracellular PHB depolymerase in Ralstonia pickettii T1 was identified. The positions of the catalytic triad (Ser190-Asp266-His330) and oxyanion hole (His108) in the amino acid sequence of PhaZd deduced from the nucleotide sequence roughly accorded with those of the extracellular PHB depolymerase of R. pickettii T1, but a signal peptide, a linker domain, and a substrate binding domain were missing. The PhaZd gene was cloned and the gene product was purified from Escherichia coli. The specific activity of PhaZd toward artificial amorphous PHB granules was significantly greater than that of other known intracellular PHB depolymerase or 3-hydroxybutyrate (3HB) oligomer hydrolases of W. eutropha H16. The enzyme degraded artificial amorphous PHB granules and mainly released various 3-hydroxybutyrate oligomers. PhaZd distributed nearly equally between PHB inclusion bodies and the cytosolic fraction. The amount of PHB was greater in phaZd deletion mutant cells than the wild-type cells under various culture conditions. These results indicate that PhaZd is a novel intracellular PHB depolymerase which participates in the mobilization of PHB in W. eutropha H16 along with other PHB depolymerases.     

Removal of endotoxin during purification of poly(3-hydroxybutyrate) from gram-negative bacteria.

Poly(3-hydroxybutyrate) (PHB) was produced by cultivating several gram-negative bacteria, including Ralstonia eutropha, Alcaligenes latus, and recombinant Escherichia coli. PHB was recovered from these bacteria by two different methods, and the endotoxin levels were determined. When PHB was recovered by the chloroform extraction method, the endotoxin level was less than 10 endotoxin units (EU) per g of PHB irrespective of the bacterial strains employed and the PHB content in the cell. The NaOH digestion method, which was particularly effective for the recovery of PHB from recombinant E. coli, was also examined for endotoxin removal. The endotoxin level present in PHB recovered by 0.2 N NaOH digestion for 1 h at 30 degrees C was higher than 10(4) EU/g of PHB. Increasing the digestion time or NaOH concentration reduced the endotoxin level to less than 1 EU/g of PHB. It was concluded that PHB with a low endotoxin level, which can be used for various biomedical applications, could be produced by chloroform extraction. Furthermore, PHB with a much lower endotoxin level could be produced from recombinant E. coli by simple NaOH digestion.     

Intracellular degradation of poly(3-hydroxybutyrate) granules of Zoogloea ramigera I-16-M

Abstract Intracellular degradation of poly(3-hydroxybutyrate) (PHB) in bacteria is not yet clear. The properties of the autodigestion of native PHB granules from Zooglea ramigera I-16-M were examined. The release of d (−)-3-hydroxybutyrate was observed only at pH values higher than about 8.5 and at relatively high ionic strength (optimal concentration 200 mM NaCl). Triton X-100 and diisopropylfluorophosphate inhibited this reaction. Addition of the supernatant fraction of Z. ramigera did not increase the release of d (−)-3-hydroxybutyrate from the native PHB granules. On the other hand, using the protease-treated PHB granules from Alcaligenes eutrophus as a substrate, PHB depolymerase activity was detected in the supernatant fraction of Z. ramigera cells. The soluble PHB depolymerase showed similar properties to the enzyme in the PHB granules. Since PHB depolymerase activity was found in fractions containing d (−)-3-hydroxybutyrate oligomer hydrolase activity, which were separated by DEAE-Toyopearl or by Sephacryl S-100, it is possible that the intracellular PHB depolymerase is identical to the oligomer hydrolase which has been purified already.     

Intracellular degradation of poly(3-hydroxybutyrate) granules of Zoogloea ramigera I-16-M.

Intracellular degradation of poly(3-hydroxybutyrate) (PHB) in bacteria is not yet clear. The properties of the autodigestion of native PHB granules from Zoogloea ramigera I-16-M were examined. The release of D(-)-3-hydroxybutyrate was observed only at pH values higher than about 8.5 and at relatively high ionic strength (optimal concentration 200 mM NaCl). Triton X-100 and diisopropylfluorophosphate inhibited this reaction. Addition of the supernatant fraction of Z. ramigera did not increase the release of D(-)-3-hydroxybutyrate from the native PHB granules. On the other hand, using the protease-treated PHB granules from Alcaligenes eutrophus as a substrate, PHB depolymerase activity was detected in the supernatant fraction of Z. ramigera cells. The soluble PHB depolymerase showed similar properties to the enzyme in the PHB granules. Since PHB depolymerase activity was found in fractions containing D(-)-3-hydroxybutyrate oligomer hydrolase activity, which were separated by DEAE-Toyopearl or by Sephacryl S-100, it is possible that the intracellular PHB depolymerase is identical to the oligomer hydrolase which has been purified already.