Pneumonia Isolates Research Articles

Amplified fragment length polymorphism of Streptococcus suis strains correlates with their profile of virulence-associated genes and clinical background

Amplified fragment length polymorphism (AFLP) typing was applied to 116 Streptococcus suis isolates with different clinical backgrounds (invasive/pneumonia/carrier/human) and with known profiles of virulence-associated genes (cps1, -2, -7 and -9, as well as mrp, epf and sly). A dendrogram was generated that allowed identification of two clusters (A and C) with different subclusters (A1, A2, C1 and C2) and two heterogeneous groups of strains (B and D). For comparison, three strains from each AFLP subcluster and group were subjected to multilocus sequence typing (MLST) analysis. The closest relationship and lowest diversity were found for patterns clustering within AFLP subcluster A1, which corresponded with sequence type (ST) complex 1. Strains within subcluster A1 were mainly invasive cps1 and mrp+ epf+ (or epf*) sly+ cps2+ strains of porcine or human origin. A new finding of this study was the clustering of invasive mrp* cps9 isolates within subcluster A2. MLST analysis suggested that A2 correlates with a single ST complex (ST87). In contrast to A1 and A2, subclusters C1 and C2 contained mainly pneumonia isolates of genotype cps7 or cps2 and epf- sly-. In conclusion, this study demonstrates that AFLP allows identification of clusters of S. suis strains with clinical relevance.

Épidémiologie des infections bactériennes nosocomiales dans une unité de réanimation néonatale et pédiatrique tunisienne

Objective The authors had for aim to describe the epidemiology of nosocomial bacterial infections in the neonatal and pediatric intensive care unit of the Tunis children's hospital. Design A prospective surveillance study was made from January 2004 to December 2004. All patients remaining in the intensive care unit for more than 48 h were included. CDC criteria were applied for the diagnosis of nosocomial infections. Results 340 patients including 249 (73%) neonates were included. 22 patients presented with 22 nosocomial bacterial infections. The incidence and the density incidence rates of nosocomial bacterial infections were 6.5% and 7.8 per 1,000 patient-days, respectively. Two types of infection were found: bloodstream infections (68.2%) and pneumonias (22.7%). Bloodstream infections had an incidence and a density incidence rate of 4.4% and 15.3 per 1,000 catheter-days, respectively. Pneumonia had an incidence and a density incidence rate of 2% and 4.4 per 1,000 mechanical ventilation-days, respectively. The most frequently isolated pathogens were Gram-negative bacteria (68%) with Klebsiella pneumoniae isolates accounting for 22.7%. The most common isolate in bloodstream infections was K. Pneumoniae (26.7%), which was multiple drug–resistant in 85% of the cases, followed by Staphylococcus aureus (20%). Pseudomonas aeruginosa was the most common isolate in pneumonia (28.6%). Associated factors of nosocomial infection were invasive devices and colonization with multiple drug-resistant Gram-negative bacteria. Conclusions The major type of nosocomial bacterial infections in our unit was bloodstream infection and the majority of infections resulted from Gram-negative bacteria. Factors associated with nosocomial bacterial infections were identified in our unit.

Incidence of Klebsiella pneumonia in street foods sold in Malaysia and their characterization by antibiotic resistance, plasmid profiling, and RAPD–PCR analysis

A total of 78 samples of street foods from different states in Malaysia were examined for the presence of Klebsiella pneumonia. K. pneumonia contamination was recorded in 32% of the samples examined. The frequency of the K. pneumonia isolates containing plasmids was shown to be 48%. Susceptibility testing showed that all isolates were highly resistant to ampicillin (100%), erythromycin (100%), rifampicin (100%), streptomycin (96%) and sulfamethoxazole (80%), but susceptible to chloramphenicol. RAPD proved to be the most effective technique in discriminating the K. pneumonia isolates since dendrogram constructed using the combination of 4 primers could differentiate 25 isolates of K. pneumonia to 25 strains. This is the first report that revealed the occurrence of K. pneumonia in street foods sold in Malaysia.

In-vitro susceptibility and molecular characterisation of macrolide resistance mechanisms among Streptococcus pneumonia isolates in The Netherlands: the DUEL 2 study

In total, 881 presumptive clinical isolates of Streptococcus pneumoniae collected from throughout The Netherlands were analysed to determine their mechanisms of macrolide resistance. Isolates were identified initially by participating laboratories using their own standard identification technique, followed by determination of MICs with Etests. Only 797 isolates were confirmed as pneumococci following bile-solubility tests, lytA PCR and 16S rRNA sequencing. Of these confirmed pneumococci, 59 (7.4%) isolates were macrolide-resistant. Analysis by PCR indicated that 34 (57.6%) isolates harboured only the erm(B) gene and 16 (27.1%) only the mef gene. Three (5.1%) isolates carried both erm(B) and mef, while six (10.2%) isolates were negative for both mechanisms. Of the six negative isolates, three had a mutation in the 23S rRNA gene, and three were negative for all mechanisms tested. No isolates with the erm(A) subclass erm(TR) gene were detected. Among the 19 mef-positive isolates, 14 (73.7%) carried the mef(A) gene, and only five (26.3%) carried the mef(E) gene. No linezolid cross-resistance or multiresistance (resistance to more than two classes of antibiotics) was observed.

Transcription of quorum-sensing system genes in clinical and environmental isolates of Pseudomonas aeruginosa.

Quorum sensing (QS)-based transcriptional responses in Pseudomonas aeruginosa have been defined on the basis of increases in transcript levels of QS-controlled genes such as lasB and aprA following the hierarchical transcriptional increases of central controllers such as the lasR gene. These increases occur at high bacterial concentrations such as early-stationary-phase growth in vitro. However, the extent to which the increases occur in a variety of clinical and environmental isolates has not been determined nor is there extensive information on allelic variation in lasR genes. An analysis of the sequences of the lasR gene among 66 clinical and environmental isolates showed that 81% have a sequence either identical to that of strain PAO1 or with a silent mutation, 15% have nucleotide changes resulting in amino acid changes, and 5% have an insertion sequence in the lasR gene. Using real-time PCR to quantify transcript levels of lasR, lasB, and aprA in the early log and early stationary phases among 35 isolates from bacteremia and pneumonia cases and the environment, we found most (33 of 35) strains had increases in lasR transcripts in early stationary phase but with a very wide range of final transcript levels per cell. There was a strong correlation (r(2) = 0.84) between early-log- and early-stationary-phase transcript levels in all strains, but this finding remained true only for the 50% of strains above the median level of lasR found in early log phase. There were significant (P < 0.05) but weak-to-modest correlations of lasR transcript levels with aprA (r(2) = 0.2) and lasB (r(2) = 0.5) transcript levels, but again this correlation occurred only in the 50% of P. aeruginosa strains with the highest levels of lasR transcripts in early stationary phase. There were no differences in distribution of lasR alleles among the bacteremia, pneumonia, or environmental isolates. Overall, only about 50% of P. aeruginosa strains from clinical and environmental sources show a lasR-dependent increase in the transcription of aprA and lasB genes, indicating that for about 50% of clinical isolates this regulatory system may not play a significant role in pathogenesis.

Clinical and economic implications of antimicrobial resistance for the management of community-acquired respiratory tract infections.

Lower respiratory tract infections (RTIs), particularly community-acquired pneumonia (CAP), account for over 50 million deaths annually worldwide. They place an extensive clinical and financial burden on healthcare authorities. Upper RTIs, usually mild and non-life threatening, also incur significant healthcare costs. The rising prevalence of resistance of the major causative agents of CAP (Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis) to beta-lactam antimicrobials and newer macrolides has necessitated new strategies for appropriate antimicrobial usage. A successful clinical outcome will depend on the patient, choice of drug, and the epidemiology and resistance of the pathogen. Treatment failure will result in increased costs, particularly if hospitalization is required. Pharmacokinetic and pharmacodynamic parameters are being used increasingly to predict maximally effective therapy and optimal bacterial eradication, thus limiting the development of resistance. Antimicrobial susceptibility criteria by MIC should be dictated by the type and location of the infection. Modifying the current MIC breakpoints for penicillin so that more pneumococcal pneumonia isolates are reported appropriately as being susceptible may lead to a decrease in the use of broad-spectrum antimicrobial therapy and its associated increased costs, in favour of more narrow-spectrum therapy. Targeting the pathogen with the most effective antimicrobial in an appropriately selected patient should optimize clinical and microbiological success and, consequently, maximize response rates and economic outcomes. In addition, research efforts need to concentrate on developing new agents with low propensity to select for or induce resistance.

Antimicrobial resistance in respiratory pathogens isolated in Brazil during 1999-2000.

The in vitro antimicrobial susceptibility of the respiratory pathogens Streptococcus Pneumoniae, Hemophilus influenzae, and Moraxella catarrhalis to commonly tested and prescribed agents was investigated during 1999-2000 and compared with results obtained during a previous 1997-1998 study. Of 448 isolates of S. Pneumoniae collected and tested in 1999-2000, 77.2% were susceptible, 19.9% were intermediate, and 2.9% were resistant to penicillin, demonstrating that there were no major changes in susceptibility to penicillin from 1997-1998 (77.1% susceptible, 18.7% intermediate, 4.2% resistant). All S. Pneumoniae isolates from 1999-2000 were susceptible to levofloxacin and vancomycin and >90% were susceptible to the B-lactams (amoxicillin-clavulanate, ceftriaxone, and cefuroxime) and macrolides (axithyromycin and clarithromycin), showing that susceptibility to these agents also remained unchanged since 1997-1998. The most notable increase in resistance between the two studies was demonstrated by trimethoprim-sulfamethoxazole, which increased from 23.4% to 38.6%. Penicillin resistance correlated with resistance to B-lactams, macrolides, and trimethoprim-sulfamethoxazole in both studies. In H. influenzae, the prevalence of B-lactamase-producing isolates remained unchanged (10.6% in 1999-2000; 11.0% in 1997-1998). All H. influenzae isolates were susceptible to levofloxacine, ceftriaxone, cefuroxime, and azithromycin, and showed no change between the two studies. Trimethoprim-sulfamethoxazole resistance was present in 40.1% of isolates in 1999-2000, and in 45.2% in 1997-1998. In M. catarrhalis, the prevalence of B-lactamase-producing isolates was unchanged (97.9% in 1999-2000;98.0% in 1997-1998). The most active agents against M. catarrhalis were azithromycin (MIC(90),< or = 0.03 microg/ml) and levofloxacin (MIC(90),< or = 0.03 microg/ml). Overall, these results suggest that, in Brazil, between 1999-2000 and 1997-1998, there have been no significant changes in the susceptibility of respiratory pathogens to any of the commonly tested and prescribed agents with the exception of trimethoprim-sulfamethoxazole for S. Pneumoniae.

Experimental Studies of Chlamydiosis in Japanese Quails

Two-week-old Japanese quails were infected intratracheally with six strains of Chlamydia psittaci isolated from calf pneumonia, swine pneumonia, goat abortion, sheep abortion, kid enteritis, and calf conjunctivitis, respectively. The Japanese quails from infected and control groups were closely observed for clinical symptoms. In order to examine the gross and microscopic lesions, quails in each group were sacrificed at 2, 5, 7, 10, 14, 21 and 30 days post infection. Alterations induced by pneumonic strains were more severe than those found in abortion isolates whereas the isolate of conjunctivitis failed to induce any lesion in the quail. Chlamydia psittaci was successfully recovered from lungs, spleen and intestinal contents of sacrificed quails. Calf pneumonia and goat abortion strains could be propagated in quails for a longer period (30 days), than in swine pneumonia, sheep abortion, kid enteritis (15 days each) and calf conjunctivitis isolates (7 days). The control quails were normal and no isolation could be made from them.

Antimicrobial resistance among community-acquired pneumonia isolates in Europe: First results from the SENTRY antimicrobial surveillance program 1997

Objective: The SENTRY antimicrobial surveillance program was established to monitor the occurrence and antimicrobial susceptibility of bacterial pathogens via an international network of sentinel hospitals. Material and Methods: Microorganisms were forwarded to the reference laboratory for testing against various antimicrobial agents using broth microdilution. Twenty European hospitals referred 286 Streptococcus pneumoniae, 309 Haemophilus influenzae, and 167 Moraxella catarrhalis isolates during the first 10 months of the study, starting in April 1997. Results: Seven percent of the S. pneumoniae isolates were highly resistant to penicillin, and 21 % showed intermediate resistance. The highly resistant pneumococcal isolates came from Coimbra, Barcelona, Athens, and London, whereas the intermediate penicillin-resistant isolates were received from all participating countries. The incidence of intermediate penicillin-resistant pneumococci was lowest in Lausanne, Freiburg and Duesseldorf, London, and Utrecht and highest in southern European countries. Fifty-five percent of the penicillin-resistant S. pneumoniae were also resistant to erythromycin, and 35% to clindamycin. Sparfloxacin, trovafloxacin, levofloxacin, and vancomycin were fully active against pneumococcal isolates. Haemophilus influenzae isolates were generally highly susceptible to most of the antibiotics tested, and 92% of the M. catarrhalis isolates were resistant to penicillin. Susceptibility to cephalosporins, ciprofloxacin, levofloxacin, and rifampicin was 100%. Conclusion: Penicillin may no longer be the first-choice drug for empirical treatment of pneumococcal infections. The newer fluoroquinolones may play a role in the empirical treatment of community-acquired pneumonia.

Evaluation of the Vitek System to accurately test the susceptibility of Pseudomonas aeruginosa clinical isolates against cefepime

A total of 300 recent blood stream and pneumonia isolates of Pseudomonas aeruginosa from 32 different medical centers in the United States were used to assess the accuracy of the Vitek System (GNS-107 card) for cefepime, a new “fourth-generation” cephalosporin. The Vitek System test result was compared to the consensus of the three other methods (reference broth microdilution, disk diffusion, Etest (AB BIODISK, Solna, Sweden)), and 271 of 300 consensus category of susceptibility sets were unanimous. The Vitek System produced a 25.3% error rate (5.3% false resistance, 20.0% minor errors). A consistent trend toward higher MIC results with the Vitek System was observed that produced a 15.3 to 21.3% lower susceptible rate compared with the other susceptibility test methods. The consensus cefepime resistance rate was only 4.3% compared to 14.0% for the Vitek GNS-107 card. The error was reproducible on triplicate repeat testing. These results indicate an unacceptable rate of false resistance being produced by the Vitek System when testing cefepime against P. aeruginosa strains. Alternative methods are suggested for this broad-spectrum antipseudomonal cephalosporin.

Mycoplasma contamination of Chlamydia pneumoniae isolates.

We examined 6 C. pneumonia isolates from The American Type Culture Collection (ATCC) and 2 Finnish isolates for Mycoplasma contamination. Three of the ATCC isolates and both of the Finnish isolates were Mycoplasma-contaminated. The contaminants were characterized by means of growth in BEa and BEg media, immunoblotting, polymerase chain reaction and pulsed field gel electrophoresis. Two of the 6 ATCC isolates [ATCC VR1355 (TWAR strain 2043) and ATCC VR1356 (TWAR strain 2023)] were infected with Mycoplasma hominis and 1 isolate [ATCC VR2282 (TWAR strain TW183)] was contaminated with both Mycoplasma hominis and Mycoplasma orale, whereas 3 of the ATCC isolates [ATCC VR1310, ATCC VR1360 (TWAR strain CM-1) and ATCC 53592 (TWAR strain AR39)] were not contaminated. The Finnish C. pneumoniae isolates Kajaani 6 and Parola were found to be contaminated with M. hominis and M. orale, respectively. The contamination of C. pneumoniae stock cultures, frequently used in the microimmunofluorescence test, with human pathogens, could pose a serious problem in C. pneumoniae serology.

A comparison of serum bactericidal activity and phenotypic characteristics of bacteremic, pneumonia-causing strains, and colonizing strains of Branhamella catarrhalis

Four blood isolates, 12 pneumonia isolates, and seven colonizing isolates of Branhamella catarrhalis were compared with respect to their ability to grow in normal human serum and in convalescent serum of a patient with B. catarrhalis bacteremia. Disease-causing isolates showed seven of 16 serum-resistant strains (43 percent) compared with one of seven (13 percent) colonizing strains. Bacteremic strains were not more serum-resistant than pneumonia-causing strains. Trypsin zones of inhibition were higher for disease-causing strains. There was no correlation between source of isolation and colistin sensitivity or ability to hemagglutinate red blood cells.

Phenotypic characteristics of Branhamella catarrhalis strains

Isolates of Branhamella catarrhalis from 13 patients with pneumonia, 6 patients with tracheobronchitis, and 8 patients who were colonized with the organism were studied with respect to susceptibility to the bactericidal action of normal human serum (NHS), glass slide hemagglutination (HA) of group O human erythrocytes, beta-lactamase production, and susceptibility to selected antimicrobial agents and laboratory drugs. A total of 18 of 27 isolates were serum resistant, 22 of 27 produced HA, and 21 of 27 were beta-lactamase positive. Statistically significant correlations were found between susceptibility to NHS and susceptibility to trypsin (r = +0.47; P = 0.01) and between susceptibility to NHS and HA (r = -0.48; P = 0.009). Significant correlations were also observed among several pairs of antimicrobial drugs. Analysis of variance showed that mean ampicillin MICs correlated with isolate group (r = -0.49; P = 0.03) in that the pneumonia isolates had higher MICs. Some phenotypic characteristics appeared to be independent of each other. These data suggest that important differences exist among clinically significant B. catarrhalis strains and that these differences may be due to differences in the cell wall envelope of the organism.

Phenotypic comparison of Pseudomonas aeruginosa strains isolated from a variety of clinical sites

Pseudomonas aeruginosa elaborates a number of extracellular products which have been shown to play a role in the pathogenesis of disease caused by this organism. In this study, we showed that the host environment markedly affects the levels of exoproducts produced. We compared the phenotypes of a number of P. aeruginosa strains obtained from a variety of clinical sources, including burn wounds, skin wounds, urine, cystic fibrosis sputum, acute pneumonia sputum, and blood. The clinical isolates were examined quantitatively for levels of total protease, elastase, phospholipase C, exotoxin A, and exoenzyme S produced in vitro under defined conditions. The exoproduct levels varied significantly, depending on the site of isolation. Elevated levels of elastase were demonstrated in strains isolated from acute lung infections, phospholipase C levels were elevated in urinary tract and blood isolates, exotoxin A levels were elevated in blood isolates, and exoenzyme S levels were increased in acute pneumonia isolates. Isolates from cystic fibrosis sputum produced low amounts of virtually all of the tested exoproducts, particularly as compared with sputum isolates from acute P. aeruginosa lung infections.

Characterization of antigens from nontypable Haemophilus influenzae recognized by human bactericidal antibodies. Role of Haemophilus outer membrane proteins.

Major outer membrane antigens, proteins, and lipopolysaccharides (LPSs), from nontypable Haemophilus influenzae were characterized and examined as targets for complement-dependent human bactericidal antibodies. Outer membranes from two nontypable H. influenzae isolates that caused otitis media and pneumonia (middle ear and transtracheal aspirates) were prepared by shearing organisms in EDTA. These membranes were compared with membranes prepared independently by spheroplasting and lysozyme treatment of whole cells and found to have: similar sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of the proteins; identical densities (rho = 1.22 g/cm3); and minimal d-lactose dehydrogenase activity indicating purity from cytoplasmic membranes. Outer membranes were solubilized in an LPS-disaggregating buffer and proteins were separated from LPS by molecular sieve chromatography. The SDS-PAGE patterns of outer membrane proteins (OMPs) from the two strains differed in the major band although other prominent bands appeared similar in molecular weight. LPS prepared by hot phenol water extraction of each of the strains contained 45% (pneumonia isolate) and 60% (otitis isolate) lipid (wt/wt), 49% and 50% carbohydrate (wt/wt), respectively, and less than 1%, 3-deoxy-manno octulosonic acid. Immunoglobulin M (IgM) purified from normal human serum (NHS) plus complement was bactericidal for both strains. Purified immunoglobulin G (IgG) from NHS killed the middle ear isolate and immune convalescent IgM from the serum of the patient with pneumonia killed his isolate. NHS or convalescent serum were absorbed with OMPs and LPS (0.6-110 micrograms) from each of the strains and immune specific inhibition of bactericidal antibody activity by each antigen was determined. OMPs from the pulmonary isolate inhibited bactericidal antibody activity directed against the isolate in both NHS (1.5 microgram of antigen) and immune serum (0.75 microgram of antigen). OMPs (60 micrograms) from the ear isolate also inhibited bactericidal activity in the respective immune serum. LPSs exhibited minimal inhibition (greater than 110 micrograms). Three human sera (two normal, one immune) were selectively depleted of 80% of antibody activity against OMPs (measured by enzyme-linked immunosorbent assay) by affinity chromatography using OMPs from the pulmonary isolate coupled to a solid phase. These OMP antibody-depleted sera also showed an 88% reduction of bactericidal activity against this strain. Immunopurified antibody against OMPs eluted from the solid phase was bactericidal.

Prospectus: The Prevention of Bacteremic Pneumococcal Pneumonia

Blood, cerebrospinal fluid, and respiratory isolates from 632 adults with pneumococcal pneumonia hospitalized from 1968 through 1972 were typed. The results were correlated with the 14 types currently being tested in pneumococcal vaccines. More than 80% of the bacteremic pneumonia isolates and two thirds of respiratory isolates were "vaccine types." Sixty percent of 43 patients with bacteremic pneumonia and 55% of patients who died from their infections would theoretically have benefited from an immunization program designed to include all persons 50 years of age and older and all younger patients with recognized underlying disease. Unexplained swings in the annual rate of pneumococcal infections, siteto-site variation in the recovery of pneumococcal types, geographic and temporal differences in the distribution of types, and the selection pressure associated with the use of a limited number of vaccine types will complicate vaccine use. (<i>JAMA</i>230:404-408, 1974)

Cephapirin: In Vitro Antibacterial Spectrum

Cephapirin, a new semisynthetic cephalosporin derivative, was found to have an antibacterial spectrum similar to that of cephalothin. Staphylococcus aureus was inhibited by cephapirin concentrations of 0.09 to 12.5 mug/ml. S. epidermidis, S. viridans, S. pyogenes, and Diplococcus pneumonia isolates were inhibited by less than 1 mug/ml. The Enterococcus required a concentration of 25 mug of antibiotic per ml for inhibition. Approximately 65% of Escherichia coli, and all Klebsiella, indole-negative Proteus, and Salmonella strains tested were inhibited by the drug. Serratia, Pseudomonas, indole-positive Proteus, and Erwinia strains were highly resistant. Inoculum size was not an important factor in determining the level of sensitivity of S. aureus to cephapirin. The antibiotic does not appear to be significantly bound to serum protein. In vitro development of resistance to the drug was demonstrated with two isolates of S. aureus.