Dominique S. Blanc

Nosocomial Pneumonia in Mechanically Ventilated Patients Receiving Antacid, Ranitidine, or Sucralfate as Prophylaxis for Stress Ulcer A Randomized Controlled Trial

Intensive care patients are at risk for bleeding from stress ulcers of the upper gastrointestinal tract [1]. Despite the decline of this complication over the last two decades [2], certain patients, such as those requiring prolonged mechanical ventilation, remain at high risk and may benefit from stress ulcer prophylaxis [1, 3-5]. Over the last few years, studies have shown that agents that raise the gastric pH may promote proliferation of bacteria in the stomach, particularly gram-negative bacilli that may originate in the duodenum [6-10]. Passive esophageal reflux and microaspiration of the gastric content along the endotracheal tube may lead to the colonization of the trachea and then to pneumonia [6, 7, 10-18]. Thus, concerns have arisen that the risk for nosocomial pneumonia may outweigh the benefit of stress ulcer prophylaxis when agents raising the gastric pH are used. Sucralfate is a complex salt of sucrose sulfate and aluminum hydroxide that appears to be as effective as antacids or histamine-2 (H2) antagonists for stress ulcer prophylaxis [2, 19, 20] but by mechanisms of action that do not result in clinically relevant gastric pH modification. Several studies have documented that gastric colonization is less frequent and of a lesser magnitude in ventilated patients treated with this agent compared with antacids or H2-antagonists [8, 21-23]. However, whether this would result in a decreased risk for nosocomial pneumonia is controversial [18, 24] because a reduction was found in some [21-23, 25] but not all [17, 21-23, 25-29] comparative studies. Methodologic differences among these studies might explain these conflicting findings [18]. For example, small numbers of patients for analysis [17, 26], low risk for pneumonia in the study patients [27, 28], periods of observation that were too brief [28], insufficient dosages of the agents that raise pH [27, 29], and wide use of enteral feeding [17] might account for the absence of reduction in the incidence of pneumonia noted in some studies. On the other hand, differences in the distribution of the base-line characteristics among the patients [22, 23], the grouping of patients receiving antacids and H2-antagonists, and analysis of subgroups of patients not randomly assigned to a treatment group [21, 25] may have biased the studies in which sucralfate was associated with lower rates of pneumonia. In addition, in two of these latter studies, the reduction of pneumonia developing in patients treated with sucralfate compared with other treatment did not reach the usual 0.05 level of significance [21, 23]. Furthermore, previous studies did not distinguish between pneumonia occurring early or late after intubation. This may be important because it is likely that early-onset pneumonia may be related to the introduction of bacteria in the trachea at the time of intubation [30-32], a process that is not expected to be influenced by the type of anti-stress ulcer prophylaxis. Therefore, we compared three anti-stress ulcer prophylaxis regimens (antacid, ranitidine, and sucralfate) in a large group of ventilated patients for the occurrence of bacterial colonization, early and late-onset nosocomial pneumonia, and overt gastrointestinal bleeding. Methods Patients The Centre Hospitalier Universitaire Vaudois is a 1100-bed hospital serving both as a municipal facility and a tertiary referral center. During a 2-year period (January 1989 to January 1991), all patients admitted to the adult medical and surgical intensive care units who were receiving mechanical ventilation and had a nasogastric tube in place were eligible for the study. Exclusion criteria were as follows: active upper gastrointestinal bleeding; treatment with antacids, H2-blockers, or sucralfate during the preceding 48 hours; creatinine levels greater than 200 mol/L; esogastric surgery; cardiac surgery; or organ transplantations. Patients likely to be extubated within 24 hours were also excluded. At intubation, patients were stratified into five categories according to the following underlying conditions: trauma (surgical intensive care unit), intervention after surgery (surgical intensive care unit), cardiac disease (medical intensive care unit), pulmonary disease (medical intensive care unit), and other medical conditions [medical intensive care unit]. Randomization was done using a random permutable table to generate a random treatment list. Treatment regimens were included in opaque, sealed envelopes. The patients were assigned to one of the following anti-stress ulcer prophylactic regimens: 1) antacid, a hospital-made suspension containing 5.4% aluminum hydroxide and 1.5% magnesium hydroxide with a buffer capacity of 1.2 mEq/mL, administered every 2 hoursthe standard dose of 20 mL was doubled if the gastric pH (tested with pH-indicator strips [Merck and Co., Darmstadt, Germany] before each administration) was less than 4.0; 2) ranitidine (Zantac, Glaxo, Bern, Switzerland) administered as a continuous intravenous infusion of 150 mg/d [100 mg/d if the blood creatinine level was between 150 and 200 mol/L]; or 3) sucralfate (Ulcogant, Merck and Co., Zurich, Switzerland) administered every 4 hours as 1 gram of suspension diluted in 20 mL of sterile water. After antacid or sucralfate was administered, the nasogastric tube was flushed with 10 mL of sterile water and clamped for 30 minutes. Each prophylactic regimen was continued until extubation unless interrupted earlier for any of the following predetermined reasons: an increase of the blood creatinine level to more than 200 mol/L, removal of the nasogastric tube, moribund state, discharge from the intensive care units, or side effects likely to be related to the stress ulcer regimen. Data Collection and Definitions For all eligible patients, demographic characteristics, diagnosis, underlying diseases, physical signs, laboratory tests, and medications were recorded prospectively by one of the investigators. However, only patients eventually intubated for more than 24 hours were followed and included in the final analysis. Glasgow coma and Acute Physiology and Chronic Health Evaluation (APACHE II) scoring systems were used to assess the severity of the acute illness [33]. The adult respiratory distress syndrome was defined by the following criteria: acute bilateral diffuse pulmonary infiltrates and severe hypoxemia without evidence of cardiogenic edema [34]. Gastric aspirates were examined for the macroscopic presence of blood (coffee ground material or fresh blood). The severity of gastric hemorrhage was assessed by clinical criteria (physical signs, blood transfusion requirements, and outcome). Chest radiographs were obtained on a daily basis or more often if clinically indicated. They were interpreted by a pneumologist who had knowledge of all relevant data except for the patients stress ulcer prophylactic regimen, gastric pH, or colonization data. Criteria for the diagnosis of ventilator-associated pneumonia were predetermined and derived from those of Salata and colleagues [35]: presence of a new or progressive infiltrate on the chest radiograph consistent with pneumonia, without other obvious cause, and associated with conditions A or B or both, defined as follows. Condition A refers to any of the following findings: pleural fluid or blood culture positive for an organism also isolated in the tracheal aspirate, radiographic cavitation, or histopathologic evidence of pneumonia. Condition B includes at least two of the following: tracheal aspirates with more than 25 leukocytes per low-power field (x 100) on a Gram stain, new leukocytosis defined as a leukocyte count greater than 10 109/L with an increase of at least 25% over baseline, or body temperature greater than 38.5 C with an increase of at least 1 C above baseline. The latter two criteria were considered only when other causes for these findings were excluded. Pneumonia was considered to be caused by a pathogen when it was cultured in high counts as the sole or predominant microorganism in the tracheal aspirate culture. Using the criteria of Langer and colleagues [30], early-onset and late-onset pneumonia were diagnosed if they occurred during the first 4 days of or 4 days after the initiation of mechanical ventilation, respectively. Consequently, only patients observed for more than 4 days could be evaluated for the development of late-onset pneumonia. A second episode of pneumonia was diagnosed when it was clearly temporally distinct from the first episode and when it involved other areas of the lungs. Pneumonia was attributed to a given anti-stress ulcer prophylactic regimen if it developed during treatment or within 2 days after extubation or treatment interruption. Death was considered to be directly related to nosocomial pneumonia when it occurred during the episode and when no other major contributing cause was present. Bacteriologic Investigations and pH Measurements Gastric and tracheal aspirates and oropharyngeal swabs were obtained twice daily and cultured quantitatively (gastric juice) or semi-quantitatively in aerobic conditions. Aerobic bacteria were identified by standard microbiologic methods. Cultures for Chlamydia species, Legionella pneumophila, or Mycoplasma pneumoniae were not done. Blood or pleural fluid cultures were ordered by the primary care physician according to the clinical situation. Gastric pH was measured twice a day using a pH meter (except in 11 patients for whom values were derived only from pH-indicator strips [Merck and Co.]). A cut-off value of 4.0 for median pH was chosen for further analysis within the three treatment groups because it has been shown to be a critical value for the growth of gram-negative bacilli in the stomach [6, 7, 25]. Colonization was defined by the presence of one microorganism at a given site on at least two occasions. A patient was considered to have gastric colonization with high counts when quantitative culture of at least one speci

Biodiversity of Amoebae and Amoeba-Resisting Bacteria in a Hospital Water Network

ABSTRACT Free-living amoebae (FLA) are ubiquitous organisms that have been isolated from various domestic water systems, such as cooling towers and hospital water networks. In addition to their own pathogenicity, FLA can also act as Trojan horses and be naturally infected with amoeba-resisting bacteria (ARB) that may be involved in human infections, such as pneumonia. We investigated the biodiversity of bacteria and their amoebal hosts in a hospital water network. Using amoebal enrichment on nonnutrient agar, we isolated 15 protist strains from 200 (7.5%) samples. One thermotolerant Hartmannella vermiformis isolate harbored both Legionella pneumophila and Bradyrhizobium japonicum. By using amoebal coculture with axenic Acanthamoeba castellanii as the cellular background, we recovered at least one ARB from 45.5% of the samples. Four new ARB isolates were recovered by culture, and one of these isolates was widely present in the water network. Alphaproteobacteria (such as Rhodoplanes, Methylobacterium, Bradyrhizobium, Afipia, and Bosea) were recovered from 30.5% of the samples, mycobacteria (Mycobacterium gordonae, Mycobacterium kansasii, and Mycobacterium xenopi) were recovered from 20.5% of the samples, and Gammaproteobacteria (Legionella) were recovered from 5.5% of the samples. No Chlamydia or Chlamydia-like organisms were recovered by amoebal coculture or detected by PCR. The observed strong association between the presence of amoebae and the presence of Legionella (P < 0.001) and mycobacteria (P = 0.009) further suggests that FLA are a reservoir for these ARB and underlines the importance of considering amoebae when water control measures are designed.

Use of an Automated Multiple-Locus, Variable-Number Tandem Repeat-Based Method for Rapid and High-Throughput Genotyping of Staphylococcus aureus Isolates

ABSTRACT Fast and reliable genotyping methods that allow real-time epidemiological surveillance would be instrumental to monitoring of the spread of methicillin-resistant Staphylococcus aureus. We describe an automated variable-number tandem repeat-based method for the rapid genotyping of Staphylococcus aureus. Multiplex PCR amplifications with eight primer pairs that target gene regions with variable numbers of tandem repeats were resolved by microcapillary electrophoresis and automatically assessed by cluster analysis. This genotyping technique was evaluated for its discriminatory power and reproducibility with clinical isolates of various origins, including a panel of control strains previously characterized by several typing methods and collections from either long-term carriers or defined nosocomial outbreaks. All steps of this new procedure were developed to ensure a rapid turnaround time and moderate cost. The results obtained suggest that this rapid approach is a valuable tool for the genotyping of S. aureus isolates in real time.

Genetic Diversity and Ecological Success of Staphylococcus aureus Strains Colonizing Humans

ABSTRACT The genetic determinants and phenotypic traits which make a Staphylococcus aureus strain a successful colonizer are largely unknown. The genetic diversity and population structure of 133 S. aureus isolates from healthy, generally risk-free adult carriers were investigated using four different typing methods: multilocus sequence typing (MLST), amplified fragment length polymorphism analysis (AFLP), double-locus sequence typing (DLST), and spa typing were compared. Carriage isolates displayed great genetic diversity which could only be revealed fully by DLST. Results of AFLP and MLST were highly concordant in the delineation of genotypic clusters of closely related isolates, roughly equivalent to clonal complexes. spa typing and DLST provided considerably less phylogenetic information. The resolution of spa typing was similar to that of AFLP and inferior to that of DLST. AFLP proved to be the most universal method, combining a phylogeny-building capacity similar to that of MLST with a much higher resolution. However, it had a lower reproducibility than sequencing-based MLST, DLST, and spa typing. We found two cases of methicillin-resistant S. aureus colonization, both of which were most likely associated with employment at a health service. Of 21 genotypic clusters detected, 2 were most prevalent: cluster 45 and cluster 30 each colonized 24% of the carrier population. The number of bacteria found in nasal samples varied significantly among the clusters, but the most prevalent clusters were not particularly numerous in the nasal samples. We did not find much evidence that genotypic clusters were associated with different carrier characteristics, such as age, sex, medical conditions, or antibiotic use. This may provide empirical support for the idea that genetic clusters in bacteria are maintained in the absence of adaptation to different niches. Alternatively, carrier characteristics other than those evaluated here or factors other than human hosts may exert selective pressure maintaining genotypic clusters.

High Proportion of Wrongly Identified Methicillin-Resistant Staphylococcus aureus Carriers by Use of a Rapid Commercial PCR Assay Due to Presence of Staphylococcal Cassette Chromosome Element Lacking the mecA Gene

ABSTRACT During a 9-month period, 217 patients were newly diagnosed as methicillin-resistant Staphylococcus aureus (MRSA) carriers by using a commercial rapid PCR-based test (GeneXpert). However, no MRSA was recovered by culturing the second swab in 61 of these patients. Further analyses showed that 28 (12.9%) of the patients harbored S. aureus isolates with a staphylococcal cassette chromosome element lacking the mecA gene and were thus incorrectly determined to be MRSA carriers.

Characterization of Cell-to-Cell Signaling-Deficient Pseudomonas aeruginosa Strains Colonizing Intubated Patients

ABSTRACT Cell-to-cell signaling involving N-acyl-homoserine lactone compounds termed autoinducers (AIs) is instrumental to virulence factor production and biofilm development by Pseudomonas aeruginosa. In order to determine the importance of cell-to-cell signaling during the colonization of mechanically ventilated patients, we collected 442 P. aeruginosa pulmonary isolates from 13 patients. Phenotypic characterization showed that 81% of these isolates produced the AI-dependent virulence factors elastase, protease, and rhamnolipids. We identified nine genotypically distinct P. aeruginosa strains. Six of these strains produced AIs [N-butanoyl-homoserine lactone or N-(3-oxo-dodecanoyl)-homoserine lactone] and extracellular virulence factors (elastase, total exoprotease, rhamnolipid, hydrogen cyanide, or pyocyanin) in vitro. Three of the nine strains were defective in the production of both AIs and extracellular virulence factors. Two of these strains had mutational defects in both the lasR and rhlR genes, which encode the N-acyl-homoserine lactone-dependent transcriptional regulators LasR and RhlR, respectively. The third of these AI-deficient strains was only mutated in the lasR gene. Our observations suggest that most, but not all, strains colonizing intubated patients are able to produce virulence factors and that mutations affecting the cell-to-cell signaling circuit are preferentially located in the transcriptional regulator genes.

Changing Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus in a Small Geographic Area over an Eight-Year Period

ABSTRACT The epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) at an international level shows that most MRSA strains belong to a few pandemic clones. At the local level, a predominance of one or two clones was generally reported. However, the situation is evolving and new clones are emerging worldwide, some of them with specific biological characteristics, such as the presence of Panton-Valentine leucocidin (PVL). Understanding these changes at the local and international levels is of great importance. Our objective was to analyze the evolution of MRSA epidemiology at multiple sites on a local level (Western Switzerland) over a period of 8 years. Data were based on MRSA reports from seven sentinel laboratories and infection control programs covering different areas. Pulsed-field gel electrophoresis was used to type MRSA isolates. From 1997 to 2004, a total of 2,256 patients with MRSA were reported. Results showed the presence of four predominant clones (accounting for 86% of patients), which could be related to known international clones (Berlin, New York/Japan, Southern Germany, and Iberian clones). Within the small geographic region, the 8-year follow-up period in the different areas showed spacio-temporal differences in the relative proportions of the four clones. Other international MRSA clones, as well as clones showing genetic characteristics identical to those of community-acquired MRSA (SCCmec type IV and the presence of PVL genes), were also identified but presumably did not disseminate. Despite the worldwide predominance of a few MRSA clones, our data showed that at a local level, the epidemiology of MRSA might be different from one hospital to another. Moreover, MRSA clones were replaced by other emerging clones, suggesting a rapid change.

Genetic diversity of Pneumocystis carinii in HIV-positive and -negative patients as revealed by PCR-SSCP typing.

ObjectiveTo describe the epidemiology of severe Pneumocystis carinii pneumonia (PCP) in HIV-infected and non HIV-infected patients. MethodsBronchoalveolar lavage specimens from 212 European patients with PCP were typed using PCR–single strand conformation polymorphism analysis of four genomic regions of P. carinii f. sp. hominis. Demographic and clinical information was obtained from all patients. ResultsTwenty-three per cent of the patients were presumably infected with a single P. c. hominis type. The other patients presented with two (50%) or more (27%) types. Thirty-five genetically stable and ubiquitous P. c. hominis types were found. Their frequency ranged from 0.4% to 10% of all isolates, and up to 15% of those from a given hospital. There was no significant association between the P. c. hominis type or number of co-infecting types per patient and geographical location, year of collection, sex, age, or HIV status. No more than three patients infected with the same type were observed in the same hospital within the same 6 month period, and no epidemiological link between the cases was found. ConclusionsThe broad diversity of types observed seems to indicate that multiple sources of the pathogen co-exist. There was no evidence that in our study population inter-human transmission played a significant role in the epidemiology of P. carinii.

Importation of Acinetobacter baumannii Into a Burn Unit: A Recurrent Outbreak of Infection Associated With Widespread Environmental Contamination

A burn patient was infected with Acinetobacter baumannii on transfer to the hospital after a terrorist attack. Two patients experienced cross-infection. Environmental swab samples were negative for A. baumannii. Six months later, the bacteria reemerged in 6 patients. Environmental swab samples obtained at this time were inoculated into a minimal mineral broth, and culture results showed widespread contamination. No case of infection occurred after closure of the unit for disinfection.

Double-Locus Sequence Typing Using clfB and spa, a Fast and Simple Method for Epidemiological Typing of Methicillin-Resistant Staphylococcus aureus

ABSTRACT Sequence-based epidemiological typing of methicillin-resistant Staphylococcus aureus (MRSA) has recently been promoted because it results in unambiguous data sets that can be organized in local and global databases. The replacement of previous typing methods, such as the highly discriminatory pulsed-field gel electrophoresis (PFGE), has been attempted with various markers and typing schemes, including spa typing and multilocus sequence typing. However, despite a number of advantages, none of these methods showed convincing evidence for performance in epidemiological typing comparable to that of PFGE. By using three sets of 48 MRSA strains comprising isolates that were (i) genetically highly diverse, (ii) genetically related, and (iii) obtained from long-term carriers, we analyzed the performance of the four highly polymorphic S. aureus markers: clfA, clfB, fnbA, and spa. Typeability, discriminatory power, in vivo stability, and evolution of these markers were compared to those of PFGE. Clearly, none of the markers alone could match the discriminatory power of PFGE (63 genotypes; index of discrimination of 0.96). Instead, this could be achieved by combining markers in pairs. We showed that by using only 3′ partial sequences of approximately 500 bp, the majority of each markers discriminatory power was displayed, and using the partial sequences, the best performance was obtained with the combination of clfB and spa (57 genotypes; index of discrimination of 0.94). Genetic changes were not observed for any of the sequence markers over a period of 3 years and in the case of partial sequences for a period of more than 4 years. This is in contrast to PFGE where changes occurred after several months. The genetic differences found between isolate pairs of long-term carriers and among highly related isolates indicated clonal evolution. A typing scheme based on 500-bp 3′ partial sequences of clfB and spa is proposed.