Communication: minimum in the thermal conductivity of supercooled water: a computer simulation study.

We report the results of a computer simulation study of the thermodynamic properties and the thermal conductivity of supercooled water as a function of pressure and temperature using the TIP4P-2005 water model. The thermodynamic properties can be represented by a two-structure equation of state consistent with the presence of a liquid-liquid critical point in the supercooled region. Our simulations confirm the presence of a minimum in the thermal conductivity, not only at atmospheric pressure, as previously found for the TIP5P water model, but also at elevated pressures. This anomalous behavior of the thermal conductivity of supercooled water appears to be related to the maximum of the isothermal compressibility or the minimum of the speed of sound. However, the magnitudes of the simulated thermal conductivities are sensitive to the water model adopted and appear to be significantly larger than the experimental thermal conductivities of real water at low temperatures.

Characterization of clinical isolates of Klebsiella pneumoniae from 19 laboratories using the National Committee for Clinical Laboratory Standards extended-spectrum beta-lactamase detection methods.

Extended-spectrum beta-lactamases (ESBLs) are enzymes found in gram-negative bacilli that mediate resistance to extended-spectrum cephalosporins and aztreonam. In 1999, the National Committee for Clinical Laboratory Standards (NCCLS) published methods for screening and confirming the presence of ESBLs in Klebsiella pneumoniae, Klebsiella oxytoca, and Escherichia coli. To evaluate the confirmation protocol, we tested 139 isolates of K. pneumoniae that were sent to Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology) from 19 hospitals in 11 U.S. states. Each isolate met the NCCLS screening criteria for potential ESBL producers (ceftazidime [CAZ] or cefotaxime [CTX] MICs were > or =2 microg/ml for all isolates). Initially, 117 (84%) isolates demonstrated a clavulanic acid (CA) effect by disk diffusion (i.e., an increase in CAZ or CTX zone diameters of > or =5 mm in the presence of CA), and 114 (82%) demonstrated a CA effect by broth microdilution (reduction of CAZ or CTX MICs by > or =3 dilutions). For five isolates, a CA effect could not be determined initially by broth microdilution because of off-scale CAZ results. However, a CA effect was observed in two of these isolates by testing cefepime and cefepime plus CA. The cefoxitin MICs for 23 isolates that failed to show a CA effect by broth microdilution were > or =32 microg/ml, suggesting either the presence of an AmpC-type beta-lactamase or porin changes that could mask a CA effect. By isoelectric focusing (IEF), 7 of the 23 isolates contained a beta-lactamase with a pI of > or =8.3 suggestive of an AmpC-type beta-lactamase; 6 of the 7 isolates were shown by PCR to contain both ampC-type and bla(OXA) genes. The IEF profiles of the remaining 16 isolates showed a variety of beta-lactamase bands, all of which had pIs of < or =7.5. All 16 isolates were negative by PCR with multiple primer sets for ampC-type, bla(OXA), and bla(CTX-M) genes. In summary, 83.5% of the K. pneumoniae isolates that were identified initially as presumptive ESBL producers were positive for a CA effect, while 5.0% contained beta-lactamases that likely masked the CA effect. The remaining 11.5% of the isolates studied contained beta-lactamases that did not demonstrate a CA effect. An algorithm based on phenotypic analyses is suggested for evaluation of such isolates.

Increasing resistance to vancomycin and other glycopeptides in Staphylococcus aureus.

Strains of Staphylococcus aureus with reduced susceptibility to glycopeptides have been reported from Japan, the United States, Europe, and the Far East. Although isolates with homogeneous resistance to vancomycin (MICs = 8 microg/mL) continue to be rare, there are increasing reports of strains showing heteroresistance, often with vancomycin MICs in the 1-4 microg/mL range. Most isolates with reduced susceptibility to vancomycin appear to have developed from preexisting methicillin-resistant S. aureus infections. Many of the isolates with reduced susceptibility to glycopeptides have been associated with therapeutic failures with vancomycin. Although nosocomial spread of the vancomycin-intermediate S. aureus (VISA) strains has not been observed in U.S. hospitals, spread of VISA strains has apparently occurred in Japan. Broth microdilution tests held a full 24 hours are optimal for detecting resistance in the laboratory; however, methods for detecting heteroresistant strains are still in flux. Disk-diffusion tests, including the Stokes method, do not detect VISA strains. The Centers for Disease Control and Prevention and other groups have issued recommendations regarding appropriate infection control procedures for patients infected with these strains.

Novel carbapenem-hydrolyzing beta-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae.

A Klebsiella pneumoniae isolate showing moderate to high-level imipenem and meropenem resistance was investigated. The MICs of both drugs were 16 microg/ml. The beta-lactamase activity against imipenem and meropenem was inhibited in the presence of clavulanic acid. The strain was also resistant to extended-spectrum cephalosporins and aztreonam. Isoelectric focusing studies demonstrated three beta-lactamases, with pIs of 7.2 (SHV-29), 6.7 (KPC-1), and 5.4 (TEM-1). The presence of bla(SHV) and bla(TEM) genes was confirmed by specific PCRs and DNA sequence analysis. Transformation and conjugation studies with Escherichia coli showed that the beta-lactamase with a pI of 6.7, KPC-1 (K. pneumoniae carbapenemase-1), was encoded on an approximately 50-kb nonconjugative plasmid. The gene, bla(KPC-1), was cloned in E. coli and shown to confer resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The amino acid sequence of the novel carbapenem-hydrolyzing beta-lactamase, KPC-1, showed 45% identity to the pI 9.7 carbapenem-hydrolyzing beta-lactamase, Sme-1, from Serratia marcescens S6. Hydrolysis studies showed that purified KPC-1 hydrolyzed not only carbapenems but also penicillins, cephalosporins, and monobactams. KPC-1 had the highest affinity for meropenem. The kinetic studies also revealed that clavulanic acid and tazobactam inhibited KPC-1. An examination of the outer membrane proteins of the parent K. pneumoniae strain demonstrated that the strain does not express detectable levels of OmpK35 and OmpK37, although OmpK36 is present. We concluded that carbapenem resistance in K. pneumoniae strain 1534 is mainly due to production of a novel Bush group 2f, class A, carbapenem-hydrolyzing beta-lactamase, KPC-1, although alterations in porin expression may also play a role.

Characterization of the extended-spectrum beta-lactamase reference strain, Klebsiella pneumoniae K6 (ATCC 700603), which produces the novel enzyme SHV-18.

Klebsiella pneumoniae K6 (ATCC 700603), a clinical isolate, is resistant to ceftazidime and other oxyimino-beta-lactams. A consistent reduction in the MICs of oxyimino-beta-lactams by at least 3 twofold dilutions in the presence of clavulanic acid confirmed the utility of K. pneumoniae K6 as a quality control strain for extended-spectrum beta-lactamase (ESBL) detection. Isoelectric-focusing analysis of crude lysates of K6 demonstrated a single beta-lactamase with a pI of 7.8 and a substrate profile showing preferential hydrolysis of cefotaxime compared to ceftazidime. PCR analysis of total bacterial DNA from K6 identified the presence of a bla(SHV) gene. K6 contained two large plasmids with molecular sizes of approximately 160 and 80 kb. Hybridization of plasmid DNA with a bla(SHV)-specific probe indicated that a bla(SHV) gene was encoded on the 80-kb plasmid, which was shown to transfer resistance to ceftazidime in conjugal mating experiments with Escherichia coli HB101. DNA sequencing of this bla(SHV)-related gene revealed that it differs from bla(SHV-1) at nine nucleotides, five of which resulted in amino acid substitutions: Ile to Phe at position 8, Arg to Ser at position 43, Gly to Ala at position 238, and Glu to Lys at position 240. In addition to the production of this novel ESBL, designated SHV-18, analysis of the outer membrane proteins of K6 revealed the loss of the OmpK35 and OmpK37 porins.

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.

Evidence of interhospital transmission of extended-spectrum beta-lactam-resistant Klebsiella pneumoniae in the United States, 1986 to 1993. The National Nosocomial Infections Surveillance System.

BACKGROUND: In addition to single-hospital outbreaks, interhospital transmission of extended-spectrum beta-lactam-resistant (ESBLR) Klebsiella pneumoniae has been suspected in some reports. However, these studies lacked sufficient epidemiological information to confirm such an occurrence. METHODS: We reviewed the surveillance data reported to the National Nosocomial Infections Surveillance (NNIS) System during 1986 to 1993 for K pneumoniae isolates and their susceptibility to either ceftazidime, cefotaxime, ceftriaxone, or aztreonam. Pulsed-field gel electrophoresis (PFGE) was used to study available ESBLR K pneumoniae isolates. RESULTS: Among 8,319 K pneumoniae isolates associated with nosocomial infections, 727 (8.7%) were resistant or had intermediate-level resistance to at least one of these antibiotics. One hospital (hospital A) accounted for 321 isolates (44.2%) of ESBLR K pneumoniae. During 1986 to 1993, the percentage of K pneumoniae isolates that were ESBLR increased from 0 to 57.7% in hospital A, from 0 to 35.6% in NNIS hospitals 0 to 20 miles from hospital A (area B), and from 1.6 to 7.3% in NNIS hospitals more than 20 miles from hospital A, including hospitals located throughout the United States. Analysis of PFGE restriction profiles showed a genetic relationship between a cluster of isolates from hospital A and some isolates from one hospital in area B, and consecutive admission in these two hospitals was confirmed for two patients from whom isolates were available. CONCLUSIONS: These data provide evidence of interhospital transmission of ESBLR K pneumoniae in one region of the United States and stress the interrelationship between hospitals when trying to control antimicrobial resistance.

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.

Evaluation of Biolog for identification of members of the family Micrococcaceae.

The Biolog Identification System (Biolog, Inc., Hayward, Calif.) was challenged at two separate laboratories with 113 coded isolates, including 33 type strains of staphylococci, 5 strains of Micrococcus spp., and 1 strain of Stomatococcus mucilaginosus. Test parameters between the sites were controlled as much as possible. Discrepancies were arbitrated by using conventional biochemicals. Overall accuracies (correct to the species level) upon initial testing were 47.7 and 59.3%, respectively, at the two laboratories. After repeat testing of isolates generating "no identification" responses or errors, the overall accuracies increased to 69.0 and 74.3% at the two sites, respectively, revealing no significant difference in the final results at the two laboratories (78 of 113 versus 84 of 113; P > 0.05). Error rates were 7.1% at one site and 9.7% at the other. The Biolog is not yet accurate enough to serve as a primary method for identifying staphylococci.

Antimicrobial susceptibility of Neisseria gonorrhoeae isolates from a military population in San Diego.

Neisseria gonorrhoeae isolates were studied to determine their patterns of antimicrobial susceptibility and possible chemotherapeutic implications. Of 370 consecutive isolates, 32 (8.7%) were penicillinase-producing N. gonorrhoeae (PPNG). The remaining 338 were subjected to disk-diffusion tests, and those apparently resistant to penicillin, tetracycline, or spectinomycin were tested by an agar-dilution method. The dilution test showed that 5.4% (20/370) were penicillin-resistant, non-PPNG strains, of which 100%, 90%, and 45% were also resistant to tetracycline, cefoxitin, and erythromycin, respectively. No resistance to spectinomycin or ceftriaxone was demonstrated, although there was an association between minimum inhibitor concentrations (MICs) of penicillin of greater than or equal to 1.0 microgram/ml and increased MICs of ceftriaxone. The overall incidence of penicillin resistant isolates, including PPNG, was 14.1% (52/370). Of the 20 penicillin-resistant, non-PPNG strains, all were also resistant to tetracycline, and another 21 exhibited tetracycline resistance but were sensitive to penicillin. The in-vitro data suggested that: (1) neither penicillin, tetracycline, nor cefoxitin were acceptable drugs for routine treatment of gonorrhea in our population during the study period; (2) spectinomycin and ceftriaxone continue to demonstrate adequate in-vitro activity against N. gonorrhoeae despite increasing in-vitro resistance to penicillin; and (3) non-plasmid-mediated resistance to penicillin may predict future resistance to ceftriaxone.

High-level tetracycline resistance in Neisseria gonorrhoeae is result of acquisition of streptococcal tetM determinant.

Recently, strains of Neisseria gonorrhoeae have been isolated which are highly resistant to tetracycline (MICs of 16 to 64 micrograms/ml). This resistance was due to the acquisition of the resistance determinant tetM, a transposon-borne determinant initially found in the genus Streptococcus and more recently in Mycoplasma hominis, Ureaplasma urealyticum, and Gardnerella vaginalis. In N. gonorrhoeae, the tetM determinant was located on a 25.2-megadalton plasmid. This plasmid arose from the insertion of tetM into the 24.5-megadalton gonococcal conjugative plasmid. The tetM determinant could be transferred to suitable recipient strains of N. gonorrhoeae by both genetic transformation and conjugation.

Chromosomally mediated resistance in Neisseria gonorrhoeae in the United States: results of surveillance and reporting, 1983-1984.

Between January 1983 and October 1984, 446 cases of infection due to chromosomally mediated resistance in Neisseria gonorrhoeae (CMRNG) were reported in 23 states. Eighty percent were detected as primary penicillin or ampicillin treatment failures. Gonococcal isolates were submitted from 175 (40%) for confirmation of resistance, susceptibility testing, gonococcal strain typing using monoclonal antibodies specific for outer membrane Protein I, and auxotyping. All were typed as Protein I serogroup IB (WII/WIII), and the majority were proline or prototrophic auxotypes. All were resistant in vitro to less than 1 microgram/ml of either penicillin or tetracycline. Comparing CMRNG with penicillinase-producing Neisseria gonorrhoeae (PPNG), we found that CMRNG were significantly more resistant to tetracycline and erythromycin, but PPNG were more resistant to penicillin (P less than .01). Because of increasing reports of gonococcal resistance in the United States, improved surveillance of clinical and laboratory resistance is needed in support of control and treatment recommendations for gonorrhea.

An unusual case of penicillinase-producing Neisseria gonorrhoeae resistant to spectinomycin in California.

We report a case of gonorrhea due to a penicillinase-producing strain of Neisseria gonorrhoeae resistant to spectinomycin in a 26-year-old man who had not been out of the United States for a year-and-a-half. His sexual contact also had no recent travel out of the United States. The genital and oropharyngeal infections were successfully treated with cefoxitin (1 g im) plus probenecid (1 g orally) and trimethoprim-sulfamethoxazole (80 mg of trimethoprim and 400 mg of sulfamethoxazole). The patient took nine of the latter tablets daily for five days. The organism was a serovar IB-3, proline-requiring auxotype. The patient's isolate contained both 2.6-megadalton and 4.4-megadalton plasmids. Measurement of minimal inhibitory concentrations (MICs) of antibiotics for the isolate confirmed the penicillin resistance and showed an MIC of spectinomycin of greater than 256 micrograms/ml. The epidemiologic investigation suggested that the source of the infection was a male contact with unusual clinical features, including bloody urethral discharge and a possible incubation period of 28 days.

Lectin characterization of gonococci from an outbreak caused by penicillin-resistant Neisseria gonorrhoeae.

A total of 40 Neisseria gonorrhoeae isolates, representing 19 penicillin-resistant isolates (from 8 heterosexual patients and 11 homosexual patients) and 21 penicillin-susceptible isolates (from 15 heterosexual patients and 6 homosexual patients) and obtained from the same geographic area, were examined. Lectin agglutination patterns were based on the reactivity of the isolates with the following 14 lectins: concanavalin A, Lens culinaris, Trichosanthes kinlowii, Griffonia simplicifolia I, Arachis hypogeae (peanut agglutinin), Glycine max (soybean agglutinin), Dolichos bifloris, Griffonia simplicifolia II, Solanum tuberosum (potato starch agglutinin), Triticum vulgaris (wheat germ agglutinin), Limax flavus, Phaseolus vulgaris, Ulex europaeus I, and Lotus tetragonolobus. All isolates were serotyped with monoclonal antibodies specific for gonococcal outer membrane protein I and auxotyped, and the plasmid content was determined. Resistant patient isolates were selected for their decreased penicillin susceptibility, and control isolates were selected for their penicillin susceptibility. Even though the patient isolates demonstrated resistance to penicillin, no phenotypic differences in lectin-grouping patterns were demonstrated between the two study groups; i.e., two predominant lectin groups were observed. No resistance-associated plasmids were detected. All patient isolates were serogroup IB (serovars IB-1, IB-2, and IB-4), whereas 12 of 21 control isolates were serogroup IA (P less than 0.05). Isolates obtained from different anatomical sites in the same patient (cervical and rectal) agreed with regard to lectin patterns and serovars but not auxotypes.

Characterization of concatemeric plasmids of Neisseria gonorrhoeae.

Three strains of Neisseria gonorrhoeae carried novel plasmids of 7.8 megadaltons (mdal) molecular mass in addition to plasmids previously observed in this organism. The presence of the 7.8-mdal plasmids was not accompanied by any distinguishable phenotype in the strain possessing them. Analysis of plasmid DNA with restriction endonucleases showed that these plasmids were composed of three directly repeated copies of a 2.6-mdal cryptic plasmid frequently found in N. gonorrhoeae. In addition, the 7.8-mdal plasmids exhibited characteristics common to the 2.6-mdal plasmid, structural lability and sites resistant to cleavage with HpaII. The concatemeric forms of the cryptic plasmid appear to be stable in these strains and do not undergo internal recombination to produce the 2.6-mdal monomer, nor were higher concatemers detected.