In vitro evaluation of different antimicrobial combinations against carbapenemase-producing Klebsiella pneumoniae: the activity of the double-carbapenem regimen is related to meropenem MIC value.

Objectives and methods: We evaluated the in vitro activity of different antimicrobial combinations with and without colistin against 39 carbapenem-resistant Klebsiella pneumoniae (CR-Kp) strains (colistin + meropenem/doripenem, colistin + tigecycline, colistin + rifampicin, gentamicin + meropenem, gentamicin + tigecycline and the double-carbapenem regimen meropenem + ertapenem) using the chequerboard method. The triple combination colistin + meropenem + tigecycline was also tested. In addition, killing studies were performed for meropenem + ertapenem. Results: Gentamicin-based combinations showed a high level of synergy. Meropenem + ertapenem was synergic in 12/39 (30.7%) of the strains, whereas based on killing studies 1 × MIC meropenem + 1 × MIC ertapenem and 2 × MIC meropenem + 1 × MIC ertapenem combinations were bactericidal and synergic at 24 h [mean area under the bactericidal curve (AUBC) 54.9 ±âŸ26.1 and 44.2 ±âŸ15.3 compared with 1 × MIC meropenem (134.5 ±âŸ40.1) and 2 × MIC meropenem (126.4 ±âŸ5.4), respectively, P < 0.0001]. When the results were stratified according to meropenem MIC, we found that the degree of synergy significantly increased for isolates with lower meropenem (and not ertapenem) MICs, up to an MIC of 128 mg/L. Among colistin-containing combinations, synergy was observed in 18/39 (46.1%), 33/34 (97%), 24/39 (61.5%) and 17/39 (43.5%) of the strains for colistin + meropenem, colistin + rifampicin, colistin + tigecycline and colistin + doripenem, respectively, including colistin-resistant strains. Colistin + meropenem + tigecycline at subinhibitory concentrations resulted in the absence of growth of 37/39 strains (94.8%). Conclusions: Our in vitro data suggest that colistin might be a valid therapeutic option against CR-Kp, even in the presence of colistin resistance, whereas the double-carbapenem regimen represents a viable option when colistin is not recommended, especially if the meropenem MIC is ≤ 128 mg/L. Since traditional antimicrobial susceptibility reports are not sufficiently informative for clinicians, synergy testing as well as actual meropenem MIC evaluation should always be performed in the case of CR-Kp infections.

Bactericidal and synergistic activity of double-carbapenem regimen for infections caused by carbapenemase-producing Klebsiella pneumoniae.

Available therapeutic options against carbapenem-resistant Klebsiella pneumoniae (CR-Kp) are limited because of the high level of resistance to other antimicrobial classes including polymyxins. The double-carbapenem regimen has been recently considered a possible therapeutic strategy. In the present study, we evaluated the in vitro bactericidal and synergistic activity of a double-carbapenem regimen consisting of ertapenem plus high-dose meropenem in a series of patients with healthcare-associated CR-Kp infections in whom the use of colistin was not indicated because of potential nephrotoxicity and/or resistance. In vitro synergy was evaluated using checkerboard and killing studies. A total of 15 patients were included in the study, with sepsis, severe sepsis and septic shock found in two (13.3%), five (33.3%) and one (6.7%) patients, respectively. Overall, the clinical/microbiological response was 12/15 (80%). Synergy was observed in 11/14 (78.6%) isolates using the checkerboard method whereas in killing studies 12/14 (85.7%) and 14/14 (100%) strains were synergistic and bactericidal at 24 h at concentrations of 1 × MIC MEM+1 × MIC ERT and 2 × MEM+1 × MIC ERT, respectively, with a significant decrease of log CFU/mL compared with other combinations (p <0.0001). The double-carbapenem regimen showed clinical and in vitro effectiveness in patients with CR-Kp infections.

24-hour blood pressure profile in Addison's disease.

The purpose of this study was to evaluate the circadian blood pressure rhythmicity in patients with primary adrenal insufficiency. Ten newly diagnosed and untreated patients with Addison's disease, 17 addisonian patients (including the previous 10) following an adequate regimen of corticosteroid replacement therapy, and 15 healthy subjects as a control group were studied. Twenty-four-hour ambulatory blood pressure and heart rate were measured automatically every 30 min using a Takeda TM-2420 recorder. The runs test, used for detecting blood pressure diurnal rhythm, was compatible with a rhythm in 2 of 10 patients with untreated Addison's disease, in 13 of 17 patients with treated Addison's disease, and in 13 of 15 normals. Six of eight addisonian patients showing no evidence of blood pressure rhythm in the untreated state acquired circadian periodicity during therapy. An analysis of blood pressure readings by Fourier series with four harmonics showed that blood pressure mesor was lower in untreated than in treated addisonian and normal subjects (P < .05). The nocturnal fall was smaller for systolic and diastolic blood pressure in untreated than in treated addisonian and normal subjects (P < .05). Adrenocortical insufficiency is often characterized by loss of circadian blood pressure rhythm, and normal rhythm can be reestablished by replacement therapy. Lack of cortisol rhythm or persistent activation of the renin-angiotensin system and sympathetic tone may play a role in this phenomenon.

Two-step (culture and PCR) diagnostic approach for differentiation of non-T. foetus trichomonads from genitalia of virgin beef bulls in Argentina.

Preputial fluids from 567 virgin Angus and Hereford bulls, 1-2 years old, were inoculated into Sutherland medium, and approximately 8.4% produced cultures with a protozoan suggestive of Tritrichomonas foetus. Under brightfield microscopy, large numbers of single-celled motile organisms with multiple anterior flagellae, a posterior flagellum, axostyle, and a visible undulating membrane were detectable. Motility was jerky and rolling, as described for T. foetus. Air-dried smears of cultures stained with Giemsa or Diff-Quick + iodine revealed an organism similar to T. foetus, although somewhat more rounded. Several organisms appeared to have four anterior flagellae. Scanning electron microscopy (5000x) of representative samples revealed four anterior flagellae on most organisms, and an axostyle that was consistently longer than that seen in T. foetus. Using pan-trichomonal primers and T. foetus-specific primers in a polymerase chain reaction (PCR) assay, amplification products of 372bp were detected in all virgin bull isolates, but only with the pan-trichomonal primers. Positive control isolates of T. foetus yielded amplification products of the expected size (372 and 347bp) with the two sets of primers, respectively. We conclude that these protozoa are not T. foetus, and note the similarity of these findings with those reported earlier in North American beef cattle. Because in several countries there is no legal treatment for bovine trichomonosis, veterinarians recommend slaughter of bulls with positive preputial cultures. The existence of easily mis-identified non-T. foetus trichomonads in the bovine prepuce suggests that the current "gold standard" diagnostic test (culture of preputial scrapings or washings) should be augmented with a more specific confirming test, such as the PCR employed in this study.

Lectin-binding sites in uterus and oviduct of normal and Campylobacter fetus subspecies venerealis-infected heifers.

This study was carried out to investigate the effect of infection with Campylobacter fetus subsp. venerealis (Cfv) on the pattern of lectin binding in the uterus and oviduct of heifers. Cfv persistence was demonstrated by bacterial isolation and immunofluorescence. Infected animals showed variations in the lectin binding pattern when compared with control animals. Cfv-infected heifers showed an increased expression of galactose and N-acetyl-galactosamine in the endometrial glands (PNA and SBA binding, respectively). The oviductal epithelium of infected heifers was strongly positive for Con A, which indicated the presence of alpha-D-mannose and alpha-D-glucose. The results of this study showed that Cfv-infection modifies the lectin binding pattern in the reproductive system of heifers. Modifications in glycoconjugates may be involved in failures of fertility and/or implantation.

Lectin histochemical study on the reproductive tract in normal and Brucella ovis-infected rams.

Histochemical studies on tissue sections showed alterations of lectin-binding reactivities in the epididymis, seminal vesicle and ampulla of Brucella ovis-infected rams. These modifications in the carbohydrate composition of organs participating in maturation, transport, and storage of spermatozoa, could be involved in the impaired fertility observed in this disease.

Aetiology of bovine abortion in Argentina.

Necropsies were performed on 354 fetuses from dairy and beef herds submitted from 1994 to 2000 to the diagnostic laboratories at Instituto Nacional de Tecnología Agropecuaria, Balcarce, Argentina. Samples from the fetuses were examined for pathogenic organisms and processed for microscopic examination. An aetiological diagnosis was made for 161 (45.5%) of the fetuses. No diagnosis was made for 193 (54.5%) fetuses. Infectious agents were isolated from 122 (34.4%) of the fetuses, bacterial agents being involved in 80 (22.6%) of these. The most common bacterial agents isolated from the fetuses were Brucella abortus in 28 fetuses, Campylobacter fetus in 26 cases, and Escherichia coli in 9 cases. Bovine herpesvirus and bovine viral diarrhoea virus were found in 9 and 6 cases, respectively. Neospora caninum was detected by an immunohistochemical technique in 26 cases (7.3%). Congenital abnormalities, dystocia and mummifications were found in 8, 19 and 11 cases, respectively.

[Characterization of strains of Escherichia coli isolated from calves with neonatal diarrhea]. / Caracterización de cepas de Escherichia coli aisladas de terneros afectados de diarrea neonatal.

Thirty fecal samples of diarrheic calves from a beef herd with previous history of neonatal diarrhea were cultured for isolation of Escherichia coli K99 (+) and for enterotoxigenic tests. The age distribution of sampled calves was: 1 animal less than 6 days old, 21 between 7-15 days old and 8 between 16-30 days old. Although most strains were classified as Isaacson et al (16) biotype four, they were negative for detection of K99 antigen by slide agglutination test. The assay for heat-stable (ST) and heat-labile (LT) enterotoxins by intragastric infant mouse inoculation and ELISA tests, respectively, were negative. More than 93% of the E. coli strains were sensitive to ampicillin, colistin, gentamycin and nitrofurantoin .

[Evaluation of 2 hemoculture media for the isolation of Brucella spp.]]. / Evaluación de dos medios de hemocultivo para el aislamiento de Brucella spp.

The diagnostic efficiency of two hemoculture media for the detection of different species of Brucella strains was evaluated. Strains of Brucella melitensis, Brucella suis, Brucella abortus, Brucella ovis, and Brucella abortus S19 were used. Each strain was diluted in phosphate buffer saline (PBS) to obtain a concentration of 10(5) colony forming units/ml (CFU/ml). Blood from goats, pigs, cattle, and sheep was mixed with the bacterial suspension to obtain a final concentration minor or equal to 10(3) CFU/ml. These blood samples were inoculated into the following media: (i) Hemobrucella (HB), (ii) Tryptose citrated broth 2% (CTB), and (iii) Controls without blood for B. melitensis and B.suis. Subculture in dishes and CFU/ml counts were made at the 1st, 3rd, 8th, 10th, 20th, and 30th post-inoculation (PI) day. Best results were obtained in the HB medium for all strains, except for B. suis, which due to the presence of a contaminant did not reach its maximum development in this medium. All strains were recovered from both media at 24 h PI, except B. ovis that was isolated from HB at 72 h PI and was not recovered from CTB. All strains remained viable for a shorter period in CTB. Under the proposed experimental conditions the HB medium was more sensitive than CTB. Future experiments should evaluate the utility of this commercial medium in clinical cases of animal brucellosis.

Humoral immune response to infectious agents in aborted bovine fetuses in Argentina.

The purpose of this study was to characterize the exposure of bovine aborted fetuses from beef and dairy herds of the humid pampas of Argentina to different infectious agents by the evaluation of fetal fluid antibodies. Presence of fetal antibodies to bovine viral diarrhea virus genotype 1 (BVDV-1), bovine herpes virus type 1 (BHV-1), Leptospira interrogans, Brucella abortus, and Neospora caninum was determined. Of the 95 fetuses processed, 66 came from 49 beef herds and 29 from 12 dairy herds. The average gestational age of the aborted fetuses was 7.1 months. Antibodies to the mentioned agents were detected in 65 of the 95 fetal fluids (68.4%). In addition, antibodies to more than one infectious agent were detected in 32 fetuses (33.7%), suggesting fetal exposure to multiple antigens during gestation. There were antibodies to BVDV-1, BHV-1, N. caninum and Leptospira interrogans in 43 (45.2%), 29 (30.5%), 26 (27.4%) and 5 (5.2%) specimens, respectively. Antibodies to B. abortus were not detected in any of the fetal fluids. The results of this study provide information on the determination of antibodies in fluids from bovine aborted fetuses exposed to different infectious agents in the region.

Influence of ursodeoxycholic acid on biliary lipids.

The advent of bile acid therapy has shed some light on the mechanisms involved in determining bile lipid secretion. The administration of cholelytic bile acids results in a lowering of cholesterol percent molar and saturation index due to a reduction in cholesterol secretion. Studies carried out after administration of bile acids showed initially that biliary cholesterol secretion rates were dependent on the hydrophobic/hydrophilic balance of the prevailing bile acid present in bile. However, more detailed investigations showed that some bile acids (cholic and chenodeoxycholic acids) did not follow this rule because of the presence of other mechanisms involved in determining biliary cholesterol secretion and a possible link between cholesterol synthesis and biliary cholesterol secretion. Several different human models have been used in more recent studies to arrive at a better understanding of the mechanisms involved in determining bile lipid secretion: obese patients, obese patients in rapid weight loss, patients with non-familial hypercholesterolemia and primary biliary cirrhosis. The findings in these studies indicate how modifications in biliary lipid secretion can easily be induced when there are changes in the relative amounts of bile acids. These changes may bring about modifications in intestinal absorption, liver synthesis, and secretion of cholesterol and bile acids that could possibly lead to the formation of lithogenic bile and subsequently to cholesterol gallstones.

Persistent elevation of serum CA 19-9 with no evidence of malignant disease.

BACKGROUND: Serum CA 19-9 is the mainstay marker for the diagnosis of biliopancreatic malignancies, though a persistent elevation can also be observed in various benign diseases. AIMS: In this study, a marked increase of serum CA 19-9 was seen in 10 patients who had no evidence of malignant disease. The possible causes of this finding are discussed. PATIENTS: Nine women and one man were studied, whose admitting diagnoses were as follows: pulmonary fibrosis in two, diabetes in two, non-ulcer dyspepsia in two, obesity in one, acute diarrhoea in one, colon diverticula in one and gastric ulcer in one. METHODS: Routine blood tests, tumour marker determinations, imaging studies and endoscopy were carried out at admission. RESULTS: Serum CA 19-9 levels ranged from 112 to 1338 IU/ml (mean 517 IU/ml). Abdominal ultrasonography, CT-scan, upper gastrointestinal X-ray series and gastrointestinal endoscopies were negative for malignancy. During the follow-up period (range 2-7 years) serum CA 19-9 values were persistently elevated in all patients. CONCLUSIONS: Our study shows that persistent and significant elevation of serum CA 19-9 can be found in non-malignant and non-cholestatic disease.

HTLV-I and HIV-I antibody frequency in thalassemics and drug addicts in southeast Sicily.

HTLV-I (Human T-Lymphotropic Virus Type 1) and HIV-1 (Human Immunodeficiency Virus Type 1) antibodies have been investigated in 702 sera samples from: 259 drug addicts nerve screened for HIV-1 (Group I), 43 drug addicts already identified as HIV-1 seropositive (Group II), 222 thalassemics (Group III) and 178 occasional blood donors (Group IV). The sera, collected from June 1984 to April 1989, were screened a first time with immunoenzymatic test and the samples proving positive were then confirmed by Western blot test. The seropositive frequency with Western blot for anti-HTLV I was 2.7% in Group I and 6.9% in Group II. Of the thalassemics, five of whom had been previously identified as seropositive for HIV-1, and of the blood donors, none proved to be seropositive for HTLV-I antibodies. With regard to HIV-1, a positive antibody response to the Western blot was found in 19.9% of the Group I subjects. Contemporaneous presence of HTLV-I and HIV-1 antibodies was found in five subjects.

Effect of two commercial vaccines to Campylobacter fetus subspecies on heifers naturally challenged.

Efficacy of two commercial vaccines containing Campylobacter fetus subspecies on heifers naturally challenged by service with an infected bull was tested. Sixteen heifers were vaccinated parentally two times with 3 weeks as interval, eight with commercial vaccine A and the other eight with commercial vaccine B. Eight other heifers were used as unvaccinated controls. Forty days after the first vaccine dose, the heifers were served by an infected bull during 60 days. Measure of systemic immune response and identification of the microorganism from genital secretions by culture and immunofluorescent antibody test (IFAT) were done. Vaccinated and control heifers had a poor reproductive performance (pregnancy rates were 2/8, 3/8 and 0/8 in groups A, B and C, respectively) and were infected by both methods during breeding time and after it. Moreover, one heifer in the groups B and C remained infected until 300 days post-breeding time. Neither vaccinated nor control heifers had an important increment of systemic antibody level. Only, they had a slight increment of antibody level after the breeding period and it may be because of natural stimulus by the infected bull during the copula. Culture and IFAT yielded high correlation on identification of C. fetus subspecies.