Nosocomial outbreak caused by a multiresistant clone of Acinetobacter baumannii: results of the case-control and molecular epidemiologic investigations.

OBJECTIVE: To describe a nosocomial outbreak caused by multiresistant Acinetobacter baumannii. DESIGN: Descriptive and case-control study. Antibiotic susceptibilities and pulsed-field gel electrophoresis (PFGE) of genomic DNA digested with SfiI and also with ApaI were used as markers of strain identity. SETTING: A large medical school-affiliated hospital in the city of Houston, Texas. RESULTS: During a 10-week period, A baumannii was isolated from 25 patients admitted to the intensive care unit (ICU). The attack rate was 14.3 per 100 ICU admissions. Case patients were intubated more frequently and for longer periods, and had longer ICU and hospital stays (P < 0.05 for each of these characteristics). Multivariate logistic regression analysis identified the length of ICU stay and the use of third-generation cephalosporins as associated with the acquisition of A baumannii. Patients infected with A baumannii had a higher mortality rate than colonized patients and control patients (P < 0.01). Sixteen isolates recovered from these 25 patients were susceptible only to imipenem/cilastatin, and PFGE confirmed that a single clone was the cause of this outbreak. Nine isolates of A baumannii from patients infected or colonized in two other hospitals in Houston during the same period, differed from the outbreak isolates by their susceptibility to ciprofloxacin. However, PFGE results were identical, indicating unsuscepted genetic relatedness among isolates from three different hospitals. CONCLUSIONS: A baumannii is an important nosocomial opportunistic pathogen and can adversely affect the outcome of ICU patients who acquire this organism. This outbreak was caused by a single clone that was isolated concurrently from three hospitals.

Development of a system for genetic and molecular analysis of Streptococcus agalactiae.

DNA-DNA hybridisation was used to compare the genetic relation of human and bovine strains of Streptococcus agalactiae. All strains showed significant homology under very stringent hybridisation conditions. The extent of relatedness did not correlate with the serological type. It was demonstrated that the S faecalis transposon Tn916 could be inserted randomly into the S agalactiae chromosome when introduced by conjugation. The ability of Tn916 insertion to cause genetic changes in S agalactiae was confirmed by identification of a mutation in lactose and trehalose fermentation associated with acquisition of the transposon. This system should be useful in genetic analysis of the pathogenicity of S agalactiae.

Comparison of enterococcal and staphylococcal beta-lactamase plasmids.

In Staphylococcus aureus, beta-lactamase (Bla) is typically associated with transducible plasmids that also encode metal resistance or with the more recently described conjugative, gentamicin resistance plasmids. The beta-lactamase gene of Enterococcus faecalis, which has been shown to be highly homologous to bla from the S. aureus plasmid p1258, is also encoded on conjugative, gentamicin resistance plasmids. To determine if the enterococcal Bla-encoding plasmids are similar to those from staphylococci, Bla-encoding plasmids from both species were compared by restriction endonuclease digestion followed by hybridization to Bla and gentamicin resistance gene probes and to one of the enterococcal Bla-encoding plasmids. Although similarities were noted in the restriction endonuclease digestion patterns among the transducible staphylococcal plasmids (pI524, pI258, and pII147) and among the conjugative Gmr staphylococcal plasmids, neither of these restriction patterns was similar to those of the enterococcal plasmids. Although the enterococcal plasmids showed extensive similarities by hybridization, the staphylococcal plasmids showed little or no similarity to the enterococcal plasmids except for bla and the gentamicin resistance genes. Whether enterococci acquired a different type of Bla-encoding plasmid or whether bla was acquired by transposition or by homologous recombination into enterococcal plasmids is unknown.

Specific agglutination of Streptococcus agalactiae from bovine mastitis by casein components of bovine milk.

Streptococcus agalactiae strains freshly isolated from bovine mastitis cases clumped within 15 min of addition of small amounts of bovine milk to a broth culture. This reaction was not observed with isolates from human infections or bovine strains that had been maintained in the laboratory for extensive periods. Intensity of the clumping response as measured by a microtiter dilution assay was highly variable. Analysis of several single colony isolates derived from one strain indicated that the clumping phenotype was genetically unstable. The clumping reaction took place in the presence of rifampicin or chloramphenicol. Milk components that caused aggregation were heat stable, relatively insensitive to proteases, and were larger than 10,000 daltons. Purified casein also induced clumping in these strains.

Identification of a Streptococcus agalactiae protein antigen associated with bovine mastitis isolates.

Immunoblotting was used to analyze the immune response of cows to Streptococcus agalactiae. Antibody from the milk of cows immunized (via the superficial inguinal lymph node) with formalinized S. agalactiae cells or from the milk of cows with S. agalactiae mastitis reacted strongly with a group of high-molecular-weight proteinaceous antigens. The two most predominant antigenic polypeptides in this group had apparent molecular weights of 97,000 and 104,000. Because the data indicated that these two antigens, as well as several minor antigens sometimes observed in the 70- to 100-kilodalton size range, seemed to be different forms of the same protein, we refer to the entire group as Sas97/104. A monoclonal antibody that was reactive with Sas97/104 was derived and was used to purify the antigen by affinity chromatography. Whole-cell and colony blot enzyme-linked immunoassays with either the monoclonal antibody or a polyclonal serum sample raised against the affinity-purified antigen indicated that this antigen (or cross-reactive proteins with higher molecular weights) is present on the S. agalactiae strains that were freshly isolated from mastitic cows. However, the antigen was not detected in S. agalactiae of human origin, bovine strains of S. agalactiae maintained for a prolonged period in the laboratory, or other streptococci. The data are consistent with the notion that Sas97/104 is a surface antigen and does not correspond to previously described type-specific antigens of group B streptococci.

Activity of LY146032 against Enterococci with and without high-level aminoglycoside resistance, including two penicillinase-producing strains.

We tested the activity of LY146032 (LY) against 57 strains of enterococci collected from Chile, Thailand, and the United States. Some of the strains were resistant to high levels of gentamicin or streptomycin (or both), and two produced beta-lactamase (Bla+). MICs of LY ranged from 0.5 to 8 micrograms/ml, and MBCs ranged from 1 to 64 micrograms/ml. In time-kill assays, a 2 to 3 log10 killing effect was observed with LY against two Bla+ strains of Streptococcus (Enterococcus) faecalis and against three strains that were highly resistant to streptomycin and gentamicin. Synergism was demonstrated with LY and streptomycin against a Bla+ strain lacking high-level streptomycin resistance. These in vitro results suggest that LY should be studied further for possible use in treatment of enterococcal infections.

Latex agglutination for rapid identification of methicillin-resistant Staphylococcus aureus recovered from selective media.

The accuracy of combining latex agglutination with selective media for the identification of methicillin-resistant Staphylococcus aureus (MRSA) was determined. Test strains were identified by latex agglutination on blood agar, the heat-stable thermonuclease test and broth microdilution MICs of oxacillin and included 97 MRSA, 56 methicillin-susceptible Staphylococcus aureus, 52 methicillin resistant, and 49 methicillin-susceptible Staphylococcus species. Isolates were grown on trypticase-soy agar with 5% sheep red blood cells (TSAB), Mueller-Hinton agar (MHA), mannitol-salt agar (MSA), and four media designed for the selective growth of MRSA:TSAB with clindamycin and gentamicin, MHA with oxacillin, MSA with oxacillin, and lipovitellin-salt-mannitol agar (LVSM) with 1 microgram oxacillin disks applied. The mean sensitivity, specificity, and positive predictive value for the combination of latex agglutination with selective media for the identification of MRSA was 96%, 99% and 98% respectively.

Disruption of the genes encoding antigen 85A and antigen 85B of Mycobacterium tuberculosis H37Rv: effect on growth in culture and in macrophages.

The mechanism of pathogenesis of Mycobacterium tuberculosis is thought to be multifactorial. Among the putative virulence factors is the antigen 85 (Ag85) complex. This family of exported fibronectin-binding proteins consists of members Ag85A, Ag85B, and Ag85C and is most prominently represented by 85A and 85B. These proteins have recently been shown to possess mycolyl transferase activity and likely play a role in cell wall synthesis. The purpose of this study was to generate strains of M. tuberculosis deficient in expression of the principal members of this complex in order to determine their role in the pathogenesis of M. tuberculosis. Constructs of fbpA and fbpB disrupted with the kanamycin resistance marker OmegaKm and containing varying amounts of flanking gene and plasmid vector sequences were then introduced as linear fragments into H37Rv by electroporation. Southern blot and PCR analyses revealed disruption of the homologous gene locus in one fbpA::OmegaKm transformant and one fbpB::OmegaKm transformant. The fbpA::OmegaKm mutant, LAa1, resulted from a double-crossover integration event, whereas the fbpB::OmegaKm variant, LAb1, was the product of a single-crossover type event that resulted in insertion of both OmegaKm and plasmid sequences. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis confirmed that expression of the disrupted gene was not detectable in the fbpA and fbpB mutants. Analysis of growth rates demonstrated that the fbpB mutant LAb1 grew at a rate similar to that of the wild-type parent in enriched and nutrient-poor laboratory media as well as in human (THP-1) and mouse (J774.1A) macrophage-like cell lines. The fbpA mutant LAa1 grew similarly to the parent H37Rv in enriched laboratory media but exhibited little or no growth in nutrient-poor media and macrophage-like cell lines. The targeted disruption of two genes encoding mycolyl transferase and fibronectin-binding activities in M. tuberculosis will permit the systematic determination of their roles in the physiology and pathogenesis of this organism.

Comparative in vitro activity of PD 127391, a new fluoroquinolone agent, against susceptible and resistant clinical isolates of gram-positive cocci.

We examined the in vitro activity of PD 127391, an investigational fluoroquinolone antibacterial agent, against staphylococci (including methicillin-resistant Staphylococcus aureus), enterococci (including beta-lactamase-producing and highly gentamicin-resistant isolates), and streptococci. The compound was active against all organisms tested and compared favorably with antimicrobial agents routinely used to treat infections with these organisms. On the basis of MICs for 90% of the strains tested, PD 127391 was 32-fold more active against all staphylococci, 16-fold more active against methicillin-resistant S. aureus, 8-fold more active against all streptococci, and 4-fold more active against all enterococci than ciprofloxacin. PD 127391 was shown to be more active than sparfloxacin, which in turn was shown to be more active than ciprofloxacin, against these gram-positive cocci. PD 127391 shows promise for the treatment of infections with gram-positive cocci, including organisms which are resistant to other commonly used antimicrobial agents.

Latex agglutination-negative methicillin-resistant Staphylococcus aureus recovered from neonates: epidemiologic features and comparison of typing methods.

An unusual strain of methicillin-resistant Staphylococcus aureus (MRSA) was repeatedly isolated from infants in a newborn special care unit (NBSC) and a newborn intensive care unit. Between January 1989 and March 1990, approximately 100 isolates from infected or colonized infants were recovered. Surveillance cultures taken during this time revealed a 20% colonization rate, which was defined as recovery of MRSA from the nares, umbilicus, or groin. Isolates were identified as S. aureus by tube coagulase reactivity and heat-stable nuclease production but were unreactive in a latex agglutination assay. Representative isolates that were collected during the outbreak and that were found to share the latex agglutination assay-negative phenotype were compared by antibiogram (12 isolates), bacteriophage typing (20 isolates), capsular polysaccharide typing (30 isolates), and plasmid as well as chromosomal DNA analyses (20 isolates). All isolates known to be associated with the outbreak had nearly identical antibiograms and were notably susceptible to clindamycin. Staphylococcal bacteriophage typing was not useful in determining the relatedness of the isolates, since the majority were nontypeable. Plasmid pattern analysis revealed one large plasmid (approximately 100 kb) of equivalent size among the isolates. Capsular polysaccharide typing revealed that 14 of 30 isolates tested were type 5. Isolates identified in children at two other hospitals in the city which were also unreactive by the latex agglutination assay and clindamycin susceptible had plasmid and antibiogram patterns identical to those of isolates from the NBSC. Pulsed-field gel electrophoresis of restriction enzyme-digested genomic DNAs from the outbreak isolates demonstrated identical patterns which could be clearly differentiated from those of other unrelated MRSA. The strain from the NBSC is, therefore, unique and underscores the need for caution in interpreting the latex agglutination reactivities of MRSA isolates.

Mycobacterial protein HbhA binds human complement component C3.

Mycobacterium tuberculosis and Mycobacterium avium are facultative intracellular pathogens that are able to survive and replicate in mononuclear phagocytes. Human complement component C3 has previously been shown to mediate attachment and phagocytosis of these bacteria by mononuclear phagocytes. In this study, a C3 ligand affinity blot protocol was used to identify a 30-kDa C3-binding protein in M. tuberculosis and Mycobacterium smegmatis and a 31-kDa C3-binding protein in M. avium. The C3-binding proteins in M. tuberculosis and M. avium localized to the cell membrane fraction and partitioned to the detergent fraction during Triton X-114 phase partitioning. The C3-binding protein from M. tuberculosis was partially purified using a cation exchange column and was shown to bind concanavalin A. The N terminus and an internal fragment of the partially purified C3-binding protein were subjected to amino acid sequence analysis. The resulting amino acid sequences matched the M. tuberculosis heparin-binding hemagglutinin (HbhA) protein. Recombinant full-length HbhA and the C terminus of HbhA fused to maltose-binding protein, but not recombinant HbhA lacking the C-terminal region, bound human C3. Recombinant full-length HbhA coated on polystyrene beads, was found to enhance the adherence and/or phagocytosis of the coated beads to J774.A1 cells in both the presence and absence of human serum. The presence of complement-sufficient serum increased the adherence of the HbhA-coated beads to the J774.A1 cells in a C3-dependent manner. If HbhA within the bacterial cell membrane functions similarly to isolated HbhA, this protein may enhance the adherence and phagocytosis of M. tuberculosis and M. avium to mononuclear phagocytes through the binding of C3 and interaction with C3 receptors on mononuclear phagocytes.