Maria J. Pons

Acute Phase is the Only Significantly Reduced Component of the Injury Response after Laparoscopic Cholecystectomy

AbstractThe objective demonstration of improved postoperative recovery suggests that the surgical injury response induced by the laparoscopic approach is less intense than that after open surgery. Twenty-five patients diagnosed as having noncomplicated gallstones were studied prospectively. They were operated by laparoscopy (group I, n = 12) or open surgery (group II, n = 13). Analgesia requirements (p < 0.026) and postoperative stay (p < 0.001) were significantly less in group I. Cholecystectomy performed by either technical options induced a significant increase over basal values of glucose, lactate, white blood cell count, prolactin, ACTH, cortisol, interleukin 6, C-reactive protein, and PCO2. Both surgical procedures induced a significant reduction of total proteins, albumin, prealbumin, free fatty acids hemoglobin, hematocrit, and pH. There were no differences between the levels of growth hormone, insulin, glucagon, or PO2 during any of the periods studied. Comparison of the results of the two cholecystectomy techniques showed that laparoscopic cholecystectomy induced a significantly less intense acute-phase response (area under the curve) of interleukin 6 (17 ± 17 versus 47 ± 26 pg/ml × hr × 102; p < 0.003), C-reactive protein (16 ± 12 versus 35 ± 16 mg/dl × hr × 10; p < 0.004), and prealbumin (16 ± 2.7 versus 13.8 ± 2.3 mg/dl × hr × 102; p < 0.05). The surgical injury response after laparoscopic cholecystectomy is similar to that after open cholecystectomy, but the acute-phase response component is less intense. This finding may be a consequence of the reduced size of the operative wound with laparoscopic cholecystectomy.

Antimicrobial Susceptibility and Mechanisms of Resistance in Shigella and Salmonella Isolates from Children under Five Years of Age with Diarrhea in Rural Mozambique

ABSTRACT The antimicrobial susceptibility and mechanisms of resistance of 109 Shigella and 40 Salmonella isolates from children with diarrhea in southern Mozambique were assessed. The susceptibility to seven antimicrobial agents was tested by disk diffusion, and mechanisms of resistance were searched by PCR or colorimetric method. A high proportion of Shigella isolates were resistant to chloramphenicol (Chl) (52%), ampicillin (Amp) (56%), tetracycline (Tet) (66%), and trimethoprim-sulfamethoxazole (Sxt) (84%). Sixty-five percent of the isolates were multidrug resistant. Shigella flexneri isolates were more resistant than those of Shigella sonnei to Amp (66% versus 0.0%, P < 0.001) and Chl (61% versus 0.0%, P < 0.001), whereas S. sonnei isolates presented higher resistance to Tet than S. flexneri isolates (93% versus 64%, P = 0.02). Resistance among Salmonella isolates was as follows: Tet and Chl, 15% each; Sxt, 18%; and Amp, 25%. Only 3% of Salmonella isolates were resistant to nalidixic acid (Nal), and none to ciprofloxacin or ceftriaxone (Cro). Among Salmonella isolates, multiresistance was found in 23%. Among Shigella isolates, antibiotic resistance was related mainly to the presence of oxa-1-like β-lactamases for Amp, dfrA1 genes for Sxt, tetB genes for Tet, and Chl acetyltransferase (CAT) activity for Chl. Among Salmonella isolates, resistance was conferred by tem-like β-lactamases for Amp, floR genes and CAT activity for Chl, tetA genes for Tet, and dfrA1 genes for Sxt. Our data show that Shigella isolates are resistant mostly to the most available, inexpensive antibiotics by various molecular mechanisms but remain susceptible to ciprofloxacin, Cro, and Nal, which is the first line for empirical treatment of shigellosis in the country.

Development of Escherichia coli rifaximin-resistant mutants: frequency of selection and stability.

OBJECTIVES To select rifaximin-resistant mutants of Escherichia coli and to establish the frequency of mutation, cross-resistance with other antimicrobial agents and the stability of the mutants obtained. METHODS Four E. coli isolates [two enteroaggregative E. coli (EAEC) and two enterotoxigenic E. coli (ETEC)] were used to obtain rifaximin-resistant mutants. The frequency of mutation in the presence of rifaximin, rifampicin and ciprofloxacin was established by growth on plates containing serial dilutions of antibiotic above the bacterial MIC. To determine the stability of rifaximin resistance, 28 selected resistant mutants were grown for 20 consecutive cultures on antibiotic-free plates. Every 10 days, the MICs of rifaximin, chloramphenicol, nalidixic acid and ciprofloxacin were established. RESULTS The frequency of mutation in the presence of rifaximin ranged between 5.7 x 10(-7) and 1.6 x 10(-6) in the case of the ETEC isolates, and between 2.0 x 10(-8) and 9.3 x 10(-8) in the case of the EAEC isolates; the frequency of mutation in the presence of rifampicin was in the order of 10(-8) and no mutant in the presence of ciprofloxacin was obtained. Twenty-six out of 28 selected mutants exhibited resistance levels around or higher than 256 mg/L. In all cases, the resistance was stable, and no reversion towards the original parental MIC was observed. In no case was the MIC of chloramphenicol, nalidixic acid or ciprofloxacin affected. CONCLUSIONS Rifaximin has a low level of resistance selection, although it may select stable highly resistant mutants in a single step. Periodical surveillance of the levels of rifaximin resistance is required to detect the possible appearance of rifaximin-resistant clinical isolates. Further studies to characterize in-depth the mechanisms of stable resistance to rifaximin are necessary.

Antimicrobial resistance in Shigella spp. causing traveller's diarrhoea (1995–2010): A retrospective analysis

BACKGROUND Shigellosis is a global human health problem causing an important morbidity among travellers returning from tropical areas. This study was aimed to describe the evolution of antimicrobial resistance profile in Shigella spp. isolated between the years 1995-2010 in patients with travellers diarrhoea (TD) returning from tropical areas. METHODS The levels of antimicrobial resistance were tested in a total of 191 Shigella spp. isolated during the period from 1995 to 2010. RESULTS A decrease of cases of diarrhoea caused by Shigella has been observed in recent years. A wide spectrum of antibiotic resistance was observed among Shigella spp. These isolates showed high levels of resistance to tetracycline (84%), co-trimoxazole (75.5%), and ampicillin (45.5%). The resistance was low to ciprofloxacin (2.1%), azithromycin (3.9%) and furazolidone (8.4%). According to the period, in the case of ampicillin, amoxicillin plus clavulanic acid, chloramphenicol, values of resistance were significantly decreasing from 1995-2000 to 2001-2010, (62.5% vs. 28.4%, 19.8% vs. 6.6%, 23.4 vs. 10.4%, respectively). Meanwhile in nalidixic acid and tetracycline the evolution of resistance has increased over time. CONCLUSIONS A decrease in the isolation number of Shigella spp. causing TD has been observed. Differential trends in the evolution of the levels of resistance to the tested antibacterial agents have been observed.

Diagnosis of Carrion's disease by direct blood PCR in thin blood smear negative samples.

Bartonella bacilliformis is the etiologic agent of Carrions disease. This disease has two well established phases, the most relevant being the so called Oroya Fever, in which B. bacilliformis infect the erythrocytes resulting in severe anemia and transient immunosuppression, with a high lethality in the absence of adequate antibiotic treatment. The presence of B. bacilliformis was studied in 113 blood samples suspected of Carrion’s disease based on clinical criteria, despite the absence of a positive thin blood smear, by two different PCR techniques (using Bartonella-specific and universal 16S rRNA gene primers), and by bacterial culture. The specific 16S rRNA gene primers revealed the presence of 21 B. bacilliformis and 1 Bartonella elizabethae, while universal primers showed both the presence of 3 coinfections in which a concomitant pathogen was detected plus Bartonella, in addition to the presence of infections by other microorganisms such as Agrobacterium or Bacillus firmus. These data support the need to implement molecular tools to diagnose Carrion’s disease.

Niveles de resistencia a quinolonas y otros antimicrobianos en cepas de Escherichia coli comensales en niños de la zona periurbana de Lima, Perú

El objetivo principal del estudio fue establecer el nivel de resistencia a antimicrobianos en un total de 222 cepas comensales de E. coli de origen fecal, en Peru. Las frecuencias de resistencia encontrados, frente los antimicrobianos evaluados, fueron: ampicilina (62,6%), cotrimoxazol (48,6%), tetraciclina (43,0%) y cloranfenicol (15,8%). Destacan los elevados niveles de resistencia a quinolonas: 32% al acido nalidixico (NAL) y 12% a ciprofloxacino (CIP). Estos elevados niveles hacia las quinolonas en cepas comensales aisladas en ninos de esta franja de edad, realzan el uso extendido y el impacto de consumo de este tipo de antimicrobianos en la comunidad, mostrando el riesgo potencial de su perdida de utilidad en el area.

Relevant role of efflux pumps in high levels of rifaximin resistance in Escherichia coli clinical isolates

BACKGROUND Enteropathogens have shown a high level of resistance against commonly used antibacterial drugs in Peru and it is necessary to explore alternative treatments. The aim of this study was to analyse the in vitro activity of rifaximin against diarrhoeagenic and commensal Escherichia coli in children less than 2 years of age. METHODS The minimal inhibitory concentration (MIC) to rifampicin and rifaximin was determined for 210 strains in the presence and absence of phenyl-arginine-β-naphthylamide (PAβN) and the mechanisms of resistance were investigated. RESULTS The MIC levels ranged between 8 and >256 mg/litre and the predominant mechanism of resistance to rifaximin was the efflux pumps inhibited by PAβN in 95.2% of the isolates. CONCLUSIONS The present MIC values are higher than those observed in other studies. Efflux pumps inhibited by PAβN were the cause of the rifaximin resistance in the majority of cases and suggest the presence of an environmental selective pressure. Consequently, rifaximin should be used with caution in the treatment of diarrhoea in Peru.

In Vitro Development and Analysis of Escherichia coli and Shigella boydii Azithromycin–Resistant Mutants

The aim of this study was to develop and analyze in vitro azithromycin (AZM)-resistant mutants of Escherichia coli and Shigella boydii. Three clinical isolates of E. coli and one S. boydii isolated from feces samples collected from children under 5 years of age with diarrhea in Lima, Peru were inoculated onto Mueller-Hinton plates containing increasing serial dilutions of AZM ranging from their specific minimal inhibitory concentration (2 or 4 mg/l) to 64 mg/l. From these plates, 16 AZM-resistant mutants were selected to determine the stability of the resistance and the presence of cross resistance with other antibiotics. The role of Phe-Arg-β-Naphthylamide (PAβN)-inhibitible efflux pumps as well as the presence of mutations in the rplV, rplD, and rrlH (23S rRNA) genes and alterations in the outer membrane profiles were determined in these 16 mutants. The rate of mutation ranged from < 2.70×10(-10) to 2.17×10(-7) for E. coli and from < 9.58×10(-10) to 1.05×10(-8) for S. boydii. E. coli mutants showed an increase in the AZM-MIC up to sixfold with one strain achieving a MIC >256 mg/l. In contrast, S. boydii only presented increases of up to twofold in MIC levels. All the strains obtained, but one showed stable AZM resistance. In the presence of PAβN, the AZM MICs decreased to parental levels in Shigella mutants, while no MIC returned to parental levels among the E. coli mutants. No cross resistance to other classes of antibiotics was found. These results show the relevance of PAβN-inhibitible efflux pumps in the basal levels and development of AZM resistance. Further studies to characterize the remaining unidentified mechanisms of AZM resistance are needed.

Fitness and molecular mechanisms of resistance to rifaximin in in vitro selected Escherichia coli mutants.

AIMS This study sought to analyze the molecular mechanisms contributing to the development of rifaximin (Rfx) resistance in vitro in Escherichia coli. Twenty-eight Rfx-resistant mutants as well as four clinical isolates of E. coli were analyzed. The results obtained show that mutations in the rpoB gene and overexpression of Phe-Arg-β-naphthylamide (PAβN)-inhibitible efflux pump were implicated in Rfx resistance. RESULTS Amino acid substitutions at position 516 of the β-subunit of RNA polymerases were the most frequently obtained (53.6% of the mutants). The efflux pump inhibitor decreased the minimal inhibitory concentration (MIC) of 71.43% (20/28) of the mutant strains. CONCLUSIONS Mutations studied in the rpoB gene and overexpression of PAβN-inhibitible efflux pumps contribute to Rfx resistance (together or not), whereas alterations in porin levels do not seem to have a relevant role in the acquisition of Rfx resistance.

Long time survival of Bartonella bacilliformis in blood stored at 4 ºC. A risk for blood transfusions

Dear Sir, Bartonella bacilliformis is an autochthonous bacterium from rural Andean areas of Peru, Ecuador and Colombia affecting less-favoured populations causing the so-called Carrion’s disease. Carrion’s disease is a re-emerging illness that has two clinically distinct phases: an acute or haematic phase, known as Oroya Fever characterised by fever and severe anaemia that may be fatal in the absence or delay of antibiotic treatment and an eruptive or tissue phase, known as Peruvian Wart1. Additionally, in endemic areas, the presence of asymptomatic individuals has been reported in whom B. bacilliformis may be recovered or detected from blood samples1. B. bacilliformis is a fastidious low-growth microorganism, requiring blood-enriched media, controlled temperature and specific atmospheric conditions to grow. Additionally the reported infections rates are around 7–20%1. These facts limit the utility of bacterial culture as a diagnostic tool. When Oroya Fever is suspected, the most common diagnostic method in endemic Peruvian areas is the Giemsa stain of a blood smear, in which the blue-coloured extra or intra-erythrocytic bacilli or coco-bacilli can be observed. Unfortunately, in centres lacking adequate training in this diagnostic technique, the sensitivity can be as low as 36%1. The limitations of blood culture together with the potential low sensitivity of Giemsa-stained blood smears and the severity of the illness usually results in empiric antibiotic treatments based on the clinical diagnosis and government guidance for treatment of the illness1. It has been reported that other members of the genus Bartonella, such as B. henselae are able to survive more than 35 days in red blood cell units stored at 4 °C, and it has also been detected in blood donors and suspicious cases of transfusion infections have been described2. Meanwhile, B. bacilliformis, contagion by direct contact with infected blood or tissues has been reported several times either in experimental or accidental manner, including at least one case of possible post-transfusion acquisition1,3. To our knowledge, only one study from 1926 reported the ability of B. bacilliformis to survive in blood samples of experimentally infected monkeys kept at 4 °C for at least 152 days4. These results suggest that the risk of B. bacilliformis infection by transfusion may be underestimated, especially when no specific test for detection in blood donors is performed in endemic areas. Thus, the aim of this report was to assess the ability of B. bacilliformis to survive long periods of time in infected human blood. Fifty-five peripheral blood samples of patients with a clinical diagnosis of Carrion’s disease and a confirmatory positive Giemsa-stained blood smear were stored at 4 °C for a minimum of 24 months. Both after collection and the end of this storage time, the samples were cultured on Columbia Agar adding 10% of sheep blood and incubated at 28 °C for a period of 10 weeks. Every 14 days the plates were visually inspected to detect any bacterial growth. Recovered microorganisms were identified by molecular tools. Thus, DNA was extracted using a commercial kit (High Pure Template Peparation Kit, Roche Applied Science, Germany) and a fragment of 1503 bp of the 16s RNA gene was amplified as previously described5. The amplified products were sequenced (Macrogen, Seoul, Korea). The initial culture showed the growth of 11 out of 55 samples (20%) after 2–5 weeks of incubation, requiring an average of 3.9 weeks to obtain positive growth, while after 24–30 months of storage at 4 °C, 6 out of these 55 (11%) samples, all belonging to the 11 with previous positive cultures (54.5%), showed bacterial growth requiring between 4 and 10 weeks of incubation. Thus 2 out of these 6 cultures were positives after 4 weeks, while the remaining isolates were positive after 6–10 weeks of growth (Table I) requiring an average time of 6.6 weeks to grow. Thus after storage the average time needed to obtain a positive culture was 71% longer that at the time of collection. These results suggests the possibility that the rate of culture positivity will be increase if incubation time is more than 10 weeks. Finally, none of the samples in which no growth was observed at the time of collection showed any growth after storage at 4 °C. Table I Microbiological data of the samples. In all cases the microorganisms grown were morphologically compatible with B. bacilliformis and all were identified as B. bacilliformis by analysis of the 16s RNA gene. Although an intensive literature search was performed, only clear information of a report of the year 1972 about a transfusional acquisition of B. bacilliformis was found. In this report a newborn died by Oroya’s Fever after receipt of a blood transfusion from a family member living in a B. bacilliformis endemic area. Although vertical transmission has been described in different cases, the mother did not have a previous Bartonella infection3. In line with this, the present results clearly show the risk of long term survival of B. bacilliformis in infected human blood stored at 4 °C, and therefore the potential risk of a transfusion transmission of this microorganism. This potential risk is enhanced firstly because this microorganism requires a prolonged incubation period (usually more than 21 days), which exceeds the usual period of blood cultivation for detection of bacterial infections2, and secondly because of the high rates of asymptomatic infections that have been reported in some studies1. Thus, it is necessary to reinforce the screening to detect B. bacilliformis in blood banks in endemic areas, as well as in those of nearby areas due to the interchange of population with endemic ones. As a corollary our results open the door to the development of molecular diagnostic tools able to be implemented in regional reference centres which might test blood samples drawn elsewhere and transported under refrigeration conditions. Development of rapid, efficient and inexpensive techniques capable of detecting B. bacilliformis in blood samples for use in risk areas is a current need. To this end several different direct blood PCR approaches are currently under investigation by our group (unpublished data). These techniques, might allow the development of a new and useful clinical diagnostic tool and enable screening for the presence of B. bacilliformis in banked blood. In summary, the results demonstrate the ability of B. bacilliformis to survive long periods of time in blood samples stored at 4 °C, suggesting the risk of transfusion-transmitted infections and the need to implement rapid techniques to detect infections in donated blood.