Transmission of methicillin-resistant Staphylococcus aureus infection through solid organ transplantation: confirmation via whole genome sequencing.

We describe two cases of donor-derived methicillin-resistant Staphylococcus aureus (MRSA) bacteremia that developed after transplantation of organs from a common donor who died from acute MRSA endocarditis. Both recipients developed recurrent MRSA infection despite appropriate antibiotic therapy, and required prolonged hospitalization and hospital readmission. Comparison of S. aureus whole genome sequence of DNA extracted from fixed donor tissue and recipients' isolates confirmed donor-derived transmission. Current guidelines emphasize the risk posed by donors with bacteremia from multidrug-resistant organisms. This investigation suggests that, particularly in the setting of donor endocarditis, even a standard course of prophylactic antibiotics may not be sufficient to prevent donor-derived infection.

Evaluation of the impact of direct plating, broth enrichment, and specimen source on recovery and diversity of methicillin-resistant Staphylococcus aureus isolates among HIV-infected outpatients.

We compared recovery of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) from nasal and groin swab specimens of 600 HIV-infected outpatients by selective and nonselective direct plating and broth enrichment. Swabs were collected at baseline, 6-month, and 12-month visits and cultured by direct plating to mannitol salt agar (MSA) and CHROMagar MRSA (CM) and overnight broth enrichment with subculture to MSA (broth). MRSA isolates were characterized by pulsed-field gel electrophoresis (PFGE), staphylococcal cassette chromosome mec (SCCmec) typing, and PCR for the Panton-Valentine leukocidin. At each visit, 13 to 15% of patients were colonized with MRSA and 30 to 33% were colonized with methicillin-susceptible S. aureus (MSSA). Broth, CM, and MSA detected 95%, 82%, and 76% of MRSA-positive specimens, respectively. MRSA recovery was significantly higher from broth than CM (P ≤ 0.001) or MSA (P ≤ 0.001); there was no significant difference in recovery between MSA and CM. MSSA recovery also increased significantly when using broth than when using MSA (P ≤ 0.001). Among specimens collected from the groin, broth, CM, and MSA detected 88%, 54%, and 49% of the MRSA-positive isolates, respectively. Broth enrichment had a greater impact on recovery of MRSA from the groin than from the nose compared to both CM (P ≤ 0.001) and MSA (P ≤ 0.001). Overall, 19% of MRSA-colonized patients would have been missed with nasal swab specimen culture only. USA500/Iberian and USA300 were the most common MRSA strains recovered, and USA300 was more likely than other strain types to be recovered from the groin than from the nose (P = 0.05).

Quantitative analysis and molecular fingerprinting of methicillin-resistant Staphylococcus aureus nasal colonization in different patient populations: a prospective, multicenter study.

OBJECTIVES: To better understand the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) colonization or infection in different patient populations, to perform quantitative analysis of MRSA in nasal cultures, and to characterize strains using molecular fingerprinting. DESIGN: Prospective, multicenter study. SETTING: Eleven different inpatient and outpatient healthcare facilities. PARTICIPANTS: MRSA-positive inpatients identified in an active surveillance program; inpatients and outpatients receiving hemodialysis; inpatients and outpatients with human immunodeficiency virus (HIV) infection; patients requiring cardiac surgery; and elderly patients requiring long-term care. METHODS. Nasal swab samples were obtained from January 23, 2006, through July 27, 2007; MRSA strains were quantified and characterized by molecular fingerprinting. RESULTS: A total of 444 nares swab specimens yielded MRSA (geometric mean quantity, 794 CFU per swab; range, 3-15,000,000 CFU per swab). MRSA prevalence was 20% for elderly residents of long-term care facilities (25 of 125 residents), 16% for HIV-infected outpatients (78 of 494 outpatients), 15% for outpatients receiving hemodialysis (31 of 208 outpatients), 14% for inpatients receiving hemodialysis (86 of 623 inpatients), 3% for HIV-infected inpatients (5 of 161 inpatients), and 3% for inpatients requiring cardiac surgery (6 of 199 inpatients). The highest geometric mean quantity of MRSA was for inpatients requiring cardiac surgery (11,500 CFU per swab). An association was found between HIV infection and colonization with the USA300 or USA500 strain of MRSA (P < or = .001). The Brazilian clone was found for the first time in the United States. Pulsed-field gel electrophoresis patterns for 11 isolates were not compatible with known USA types or clones. CONCLUSION: Nasal swab specimens positive for MRSA had a geometric mean quantity of 794 CFU per swab, with great diversity in the quantity of MRSA at this anatomic site. Outpatient populations at high risk for MRSA carriage were elderly residents of long-term care facilities, HIV-infected outpatients, and outpatients receiving hemodialysis.

Evaluation of methods to identify the Klebsiella pneumoniae carbapenemase in Enterobacteriaceae.

The Klebsiella pneumoniae carbapenem (KPC) beta-lactamase occurs in Enterobacteriaceae and can confer resistance to all beta-lactam agents including carbapenems. The enzyme may confer low-level carbapenem resistance, and the failure of susceptibility methods to identify this resistance has been reported. Automated and nonautomated methods for carbapenem susceptibility were evaluated for identification of KPC-mediated resistance. Ertapenem was a more sensitive indicator of KPC resistance than meropenem and imipenem independently of the method used. Carbapenemase production could be confirmed with the modified Hodge test.

Characterization of erythromycin-resistant isolates of Staphylococcus aureus recovered in the United States from 1958 through 1969.

We tested 16 erythromycin-resistant clinical isolates of S. aureus, recovered from patients hospitalized in the United States from 1958 to 1969, for the presence of ermA, ermB, and ermC by using PCR. Fifteen of 16 isolates contained at least one copy of ermA; the remaining isolate, which was also clindamycin resistant, contained ermB. Eight of the 15 isolates harboring ermA, all of which were inducible, contained a single copy of the gene in the chromosome, while the remaining seven isolates had two copies of the gene. ermB was plasmid encoded and mediated constitutive resistance to erythromycin.

Detection of Tn917-like sequences within a Tn916-like conjugative transposon (Tn3872) in erythromycin-resistant isolates of Streptococcus pneumoniae.

A series of macrolide-lincosamide-streptogramin B (MLS)-resistant pneumococcal isolates of a variety of serotypes was examined and was found to contain Tn917-like elements by DNA-DNA hybridization. Like Tn1545, Tn917 also encodes an ermAM gene but does not mediate resistance to other antimicrobial agents. Furthermore, nucleotide sequence analyses of the DNAs flanking three of the Tn917-like elements revealed that they were inserted into orf9 of a Tn916-like element in a composite transposon-like structure (Tn3872). Other MLS-resistant strains appeared to contain Tn1545-like elements that had suffered a deletion of sequences including the aphA-3 sequences responsible for kanamycin resistance. Thus, the MLS resistance phenotype in pneumococci appears to be mediated by the ermAM present on a much wider variety of genetic elements than was previously appreciated.

Identification of multiple clones of extended-spectrum cephalosporin-resistant Streptococcus pneumoniae isolates in the United States.

We characterized 12 isolates of Streptococcus pneumoniae with various levels of susceptibility of penicillin and extended-spectrum cephalosporins by antimicrobial susceptibility patterns, serotypes, ribotypes, chromosomal DNA restriction patterns by pulsed-field gel electrophoresis, multilocus enzyme electrophoresis patterns, penicillin-binding protein (PBP) profiles, and DNA restriction endonuclease cleavage profiles of pbp1a, pbp2x, and pbp2b. Seven cefotaxime-resistant (MIC, > or = 2 micrograms/ml) serotype 23F isolates were related on the basis of ribotyping, pulsed-field gel electrophoresis, and multilocus enzyme electrophoresis, but they had two slightly different PBP patterns: one unique to strains for which the MIC of penicillin is high (4.0 micrograms/ml) and one unique to strains for which the MIC of penicillin is low (0.12 to 1.0 micrograms/ml). The pbp1a and pbp2x fingerprints were identical for the seven isolates; however, the pbp2b fingerprints were different. An eighth serotype 23F isolate with high-level resistance to cephalosporins was not related to the other seven isolates by typing data but was a variant of the widespread, multiresistant serotype 23F Spanish clone. The PBP profiles and fingerprints of pbp1a, pbp2x, and pbp2b were identical to those of the Spanish clone isolate. An additional serotype 6B isolate with high-level resistance to cephalosporins had unique typing profiles and was unrelated to the serotype 23F cephalosporin-resistant isolates but was related on the basis of genetic typing methods to a second serotype 6B isolate that was cephalosporin susceptible. The serotype 6B isolates had different PBP profiles and fingerprints for pbp1a, but the fingerprints for pbp2x and pbp2b were the same.

Genetic analysis of clinical isolates of Streptococcus pneumoniae with high-level resistance to expanded-spectrum cephalosporins.

Streptococcus pneumoniae CS109 and CS111 were isolated in the United States in 1991 and have high levels of resistance to expanded-spectrum cephalosporins (MICs of 8 and 32 micrograms of cefotaxime per ml, respectively). CS109, but not CS111, also showed high-level resistance to penicillin. As both strains expressed the serotype 23F capsule, were very closely related in overall genotype, and possessed identical or closely related mosaic pbp1a, pbp2x, and pbp2b genes, it is likely that they have arisen from a recent common ancestor. High-level resistance to expanded-spectrum cephalosporins was entirely due to alterations of penicillin-binding proteins (PBPs) 1a and 2x, since a mixture of the cloned pbp1a and pbp2x genes from the resistant strains could transform the susceptible strain R6 to the full level of cephalosporin resistance of the clinical isolates. Both PBP1a and PBP2x of these strains were more resistant to inhibition by cephalosporins than those of typical highly penicillin-resistant isolates. The pbp1a genes of CS109 and CS111 were identical in sequence, and the fourfold difference in their levels of resistance to cephalosporins was due to a Thr-550-->Ala substitution at the residue following the conserved Lys-Ser-Gly motif of PBP2x. This substitution was also the major cause of the 16-fold-lower resistance of CS111 to penicillin. The pbp2x gene of CS111, in an appropriate genetic background, could provide resistance to 16 micrograms of cefotaxime per ml but only to 0.12 microgram of benzylpenicillin per ml. Removal of the codon 550 mutation resulted in a pbp2x gene that provided resistance to 4 microgram of cefotaxime per ml and 4 microgram of benzylpenicillin per ml. The Thr-550-->Ala substitution in CS111 therefore appears to provide increased resistance to expanded-spectrum cephalosporins but a loss of resistance to penicillin.

A cluster of invasive pneumococcal disease in young children in child care.

OBJECTIVE: To investigate a cluster of invasive pneumococcal disease in children 8 to 26 months of age, using standard microbiological procedures and ribosomal DNA gene-restriction patterns to characterize the outbreak strain. DESIGN: Outbreak investigation. SETTING: A family child-care home with six children in Baltimore, Md. RESULTS: During an 8-day period, three of the six children in the family child-care home had febrile illnesses with pneumococcal bacteremia, and a fourth had purulent pneumococcal conjunctivitis. Type 12F Streptococcus pneumoniae was isolated from the four ill children and from the nasopharynges of the two healthy children. Ribotyping revealed all outbreak isolates had an identical ribotype pattern. Administration of rifampin to the children did not eradicate carriage of the organism. CONCLUSIONS: Our data demonstrate that child care provides an opportunity for outbreak of invasive pneumococcal disease in young children. This observation suggests a need for increased alertness for clusters of pneumococcal disease in young children in child-care facilities and underscores the necessity for a pneumococcal vaccine that is effective in infants and young children.

The spread of multiply resistant Streptococcus pneumoniae at a day care center in Ohio.

Streptococcus pneumoniae, type 23F, resistant to penicillin (MIC, 2 micrograms/mL) and multiple other antimicrobic agents, was isolated from middle ear fluid of a child with otitis media attending a day care center in Ohio. To determine the extent of spread of this strain, nasopharyngeal culture surveys were done, and 52 carriers were identified among 250 children attending the index day care center. No carriers were found among 121 children at two other day care centers in the same urban area. Use of prophylactic doses of antibiotics (P < .001) and frequent use of antibiotics (P < 0.001) were risk factors for nasopharyngeal carriage. Carriers were more likely to have had frequent otitis media episodes (P < .02) and otitis media not responsive to antimicrobial therapy (P < .001). Strategies to limit the spread of highly resistant pneumococcal strains should include encouraging judicious use of antimicrobic agents and reevaluating indications for prophylactic use of antimicrobic agents.

Analysis of multiply antimicrobial-resistant isolates of Streptococcus pneumoniae from the United States.

Streptococcus pneumoniae isolates resistant to penicillin, chloramphenicol, tetracycline and sulfamethoxazole-trimethroprim are being recovered with increasing frequency in the United States. We analyzed the penicillin-binding proteins (PBPs), multilocus enzyme electrophoresis (MLEE) genotypes, and ribotypes of 22 multiresistant serotype 23F isolates of S. pneumoniae from the United States and 1 isolate each from Spain and South Africa. Also included were seven multiresistant isolates of other serotypes, three penicillin-resistant but chloramphenicol-susceptible serotype 23F isolates, and two penicillin-susceptible isolates (one penicillin-susceptible isolate was serotype 23F). Fifteen of the 22 multiresistant isolates from the United States and the isolates from Spain and South Africa had identical PBP patterns, MLEE profiles, and ribotypes. Six of the remaining seven multiresistant isolates were related by PBP pattern, but demonstrated slightly different MLEE and/or ribotype profiles, possibly because of acquisition of additional resistance markers (four of the six isolates were also resistant to erythromycin). The remaining multiresistant serotype 23F isolate had a unique PBP pattern and ribotype and was only distantly related to the other pneumococcal isolates by MLEE analysis. The PBP patterns, MLEE profiles, and ribotypes of the multiresistant serotype 23F isolates were easily distinguished from those of six multiresistant isolates of other serotypes; three other penicillin-resistant, chloramphenicol-susceptible, serotype 23F isolates; and two penicillin-susceptible isolates. One exception was a multiresistant serotype 19A isolate that was highly related to the clonal group by PBP pattern and MLEE analysis and that had a ribotype similar to those of the other erythromycin-resistant serotype 23F isolates. MLEE analysis and ribotyping were more discriminating than were the PBP patterns in discerning strain differences. These data strongly suggest that a multiresistant clone of S. pneumoniae serotype 23F that is related to multiresistant isolates from Spain and South Africa has become disseminated in the United States. Clinicians should be alerted to the spread of these multiresistant strains in the United States.

The role of beta-lactamase in staphylococcal resistance to penicillinase-resistant penicillins and cephalosporins.

We showed that most Staphylococcus aureus strains that have borderline or intermediate susceptibility to the penicillinase-resistant penicillins (PRPs) react this way because of the activity of their beta-lactamase on these antimicrobial agents. These strains produced large amounts of staphylococcal beta-lactamase that rapidly hydrolyzed penicillin and partially hydrolyzed the PRPs. Susceptibility to hydrolysis was penicillin greater than oxacillin greater than cephalothin greater than methicillin. The borderline results and the hydrolysis could be prevented by the beta-lactamase inhibitors clavulanic acid and sulbactam. For intrinsically methicillin-resistant (heteroresistant) S. aureus, the inhibitors reduced the penicillin MICs, but the strains remained resistant to all the beta-lactam antimicrobial agents, including penicillin. We conclude that the borderline in vitro susceptibility or resistance to PRPs in most of these S. aureus strains is mediated by beta-lactamase and they are not heteroresistant or intrinsically resistant. We do not know whether this in vitro resistance is expressed clinically.

The in vitro activity of sulphones alone and in combination with ampicillin or amoxicillin against Legionella pneumophila and other Legionella spp. including beta-lactamase studies.

Penicillinate sulphone beta-lactamase inhibitors, sulbactam, and BL-P2013, were very effective alone and in combination with ampicillin-like penicillins against 34 strains of Legionellae. The minimum concentrations inhibiting 50% of tested isolates (MIC50) results were as follows: sulbactam, BL-P2013, and amoxicillin = 2.0 micrograms/ml; ampicillin = 1.0 microgram/ml; erythromycin = 0.5 microgram/ml; and rifampin = 0.03 microgram/ml. Synergy was commonly observed when the sulphones were combined with ampicillin or amoxicillin, generally reducing the drug minimum inhibitory concentrations (MICs) fourfold to eightfold (synergy rates 85-91%). BL-P2013 was a slightly more active inhibitor of Legionella spp. beta-lactamase than dicloxacillin or sulbactam.

New recommendations for disk diffusion antimicrobial susceptibility tests for methicillin-resistant (heteroresistant) staphylococci.

The agar disk diffusion susceptibility test was reevaluated for its ability to discriminate between susceptible and resistant Staphylococcus aureus (128 strains) and coagulase-negative staphylococci (19 strains) when tested with methicillin, oxacillin, and nafcillin. The results show that the current recommendations for disk potencies and interpretive zone diameters do not fit well with MIC correlates that we now recommend. Based on data from this study, we suggest that these parameters of the test be changed. For methicillin, we recommend a 10-micrograms disk with breakpoints of less than or equal to 11 mm (greater than or equal to 16 micrograms/ml) to indicate resistance and greater than or equal to 15 mm (less than or equal to 4 micrograms/ml) to indicate susceptibility. For oxacillin and nafcillin, we recommend 4-micrograms disks with breakpoints of less than or equal to 12 mm (greater than or equal to 8 micrograms/ml) to indicate resistance and greater than or equal to 16 mm (less than or equal to 2 micrograms/ml) to indicate susceptibility. MIC breakpoints were from a broth microdilution system which used a medium containing salt. If one of these three penicillins were to be selected for routine tests, we would recommend oxacillin, based on our data, but we recognize that this may depend upon the population of staphylococci within a particular hospital.

Successful use of broth microdilution in susceptibility tests for methicillin-resistant (heteroresistant) staphylococci.

We studied the broth microdilution antimicrobial susceptibility testing procedure to see whether it could be made reliable for determining resistance of staphylococci to methicillin, oxacillin, nafcillin, and cephalothin. With 45 selected strains of Staphylococcus aureus and 12 selected strains of Staphylococcus epidermidis we found that the addition of 2% NaCl to cation-supplemented Mueller-Hinton broth permitted us to discriminate reliably between resistant and susceptible organisms. A screening test in which resistant staphylococci grew on agar containing 4% NaCl and methicillin (10 micrograms/ml), oxacillin (6 micrograms/ml), or nafcillin (6 micrograms/ml) incubated at 35 degrees C for 24 h (additional 24 h if no growth) was also reliable. In vitro cephalothin resistance occurred in heteroresistant S. aureus but usually did not occur in heteroresistant S. epidermidis.

Rifampin: spectrum of antibacterial activity.

Rifampin was studied for determination of its spectrum of activity against many bacteria of clinical importance. Most of the minimum inhibitory concentrations (MICs) were determined by agar dilution but some were determined by broth microdilution. Staphylococci were the most susceptible, with mode MICs of 0.015 microgram/ml, but most streptococcal strains, except Streptococcus faecalis, had mode MICs less than or equal to 1 microgram/ml. Haemophilus influenzae, Neisseria gonorrhoeae, Neisseria meningitidis, and Listeria monocytogenes were susceptible and had mode MICs of 1, 0.25, 0.03, and less than or equal to 0.12 microgram/ml, respectively. Legionella species had geometric mean MICs ranging from 0.027 to 0.25 microgram/ml. The rapidly growing mycobacteria, Mycobacterium chelonei and Mycobacterium fortuitum, were resistant, with mode of greater than 64 micrograms/ml. Enterobacteriaceae, Acinetobacter species, and Pseudomonas species had mode MICs ranging from 4 to 64 micrograms/ml. Thus, the authors conclude that, on the basis of these in vitro data and an MIC breakpoint of less than or equal to 2 micrograms/ml, gram-positive cocci (except for some enterococci), H. influenzae, N. gonorrhoeae, N. meningitidis, Legionella, and L. monocytogenes may be clinically susceptible to rifampin.

Legionella oakridgensis: unusual new species isolated from cooling tower water.

We describe a new species of Legionella represented by 10 strains isolated from industrial cooling towers. Legionella oakridgensis differed genetically from the other seven species of Legionella in DNA hybridization studies and differed serologically in direct fluorescent-antibody tests. The new species, unlike all other species except L. jordanis, did not require added L-cysteine for growth in serial transfer on charcoal-yeast extract agar. L. oakridgensis, as well as three other species tested, required L-cysteine for primary isolation from animal tissues. L. oakridgensis was the only species of Legionella that failed to produce alkaline phosphatase at pH 8.5. In all other respects, it resembled other species of Legionella, including having a high content of branched-chain cellular fatty acids and being pathogenic for guinea pigs. These bacteria have not yet been associated with human disease, but they are potential causes of legionellosis.