Identification of 16S rRNA methylase-producing Acinetobacter baumannii clinical strains in North America.

Five highly amikacin-resistant Acinetobacter baumannii isolates were collected at a medical center in Pennsylvania. The aminoglycoside resistance was due to the production of the 16S rRNA methylase ArmA. Two of the isolates coproduced OXA-23 beta-lactamase and were highly resistant to carbapenems as well. The isolates were genetically closely related by pulsed-field gel electrophoresis.

Analysis of antibiotic resistance genes in multidrug-resistant Acinetobacter sp. isolates from military and civilian patients treated at the Walter Reed Army Medical Center.

Military medical facilities treating patients injured in Iraq and Afghanistan have identified a large number of multidrug-resistant (MDR) Acinetobacter baumannii isolates. In order to anticipate the impact of these pathogens on patient care, we analyzed the antibiotic resistance genes responsible for the MDR phenotype in Acinetobacter sp. isolates collected from patients at the Walter Reed Army Medical Center (WRAMC). Susceptibility testing, PCR amplification of the genetic determinants of resistance, and clonality were determined. Seventy-five unique patient isolates were included in this study: 53% were from bloodstream infections, 89% were resistant to at least three classes of antibiotics, and 15% were resistant to all nine antibiotics tested. Thirty-seven percent of the isolates were recovered from patients nosocomially infected or colonized at the WRAMC. Sixteen unique resistance genes or gene families and four mobile genetic elements were detected. In addition, this is the first report of bla(OXA-58)-like and bla(PER)-like genes in the U.S. MDR A. baumannii isolates with at least eight identified resistance determinants were recovered from 49 of the 75 patients. Molecular typing revealed multiple clones, with eight major clonal types being nosocomially acquired and with more than 60% of the isolates being related to three pan-European types. This report gives a "snapshot" of the complex genetic background responsible for antimicrobial resistance in Acinetobacter spp. from the WRAMC. Identifying genes associated with the MDR phenotype and defining patterns of transmission serve as a starting point for devising strategies to limit the clinical impact of these serious infections.

Detection of very high-level penicillin-resistant variants of the Tennessee (23 F)-4 clone via single and serial transformations with four serotype 19 A international pneumococcal clones.

In the United States, penicillin-resistant variants of the Tennessee (Tenn) (23 F)-4 clone account for a substantial proportion of the very-high-level penicillin-resistant (MIC 8 microg/ml) infections in the 7-valent pneumococcal protein conjugate vaccine (PCV 7) era. Serotype 19 A strains account for an increasing proportion of penicillin-nonsusceptible Streptococcus pneumoniae infections. Sequential transformations of the Tenn (23 F)-4 clone (penicillin MIC 0.1 microg/ml) were performed with four penicillin-nonsusceptible serotype 19 A international clones (penicillin MIC): S. Africa (19 A)-7 (0.5 microg/ml), Hungary (19 A)-6 (2 microg/ml), Slovakia (19 A)-11 (8 microg/ml), and South Africa (19 A)-13 (8 microg/ml). Fifty-two transformants were characterized by MICs, serogroup-specific PCR, pbp PCR restriction profile and sequence, psp A PCR restriction profile, and erm/mef PCR. A subset was analyzed with multilocus sequence typing (MLST) and pulsed-field gel electrophoresis. Serotype 23 F transformants with penicillin MIC >or= 8 microg/ml were detected through a single transformation with the Hungary (19 A)-6 clone or serial transformations using two to three different clones. Forty-four percent (14/32) of the transformants incorporated >or=1 new MLST allele. Using encapsulated donors, very-high-level penicillin resistant variants of the Tenn (23 F)-4 clone were detected. In addition to detecting stepwise increases in penicillin MIC, a 12-fold increase in penicillin MIC was achieved through a single transformation. This large increase in MIC may explain why this clone is commonly associated with very-high-level resistance in natural populations. Recombination within the MLST housekeeping genes was commonly detected in the transformants that had acquired penicillin resistance.

Erythromycin-nonsusceptible Streptococcus pneumoniae in children, 1999-2001.

After increasing from 1995 to 1999, invasive erythromycin-nonsusceptible Streptococcus pneumoniae rates per 100,000 decreased 53.6% in children from Baltimore, Maryland (US), from 1999 to 2001, which was partially attributed to strains related to the mefE-carrying England14-9 clone. The decline in infection rates was likely due to the pneumococcal 7-valent conjugate vaccine.

Acute otitis media due to penicillin-nonsusceptible Streptococcus pneumoniae before and after the introduction of the pneumococcal conjugate vaccine.

BACKGROUND: The impact of the 7-valent pneumococcal conjugate vaccine (PCV7 [Prevnar]) on penicillin-nonsusceptible Streptococcus pneumoniae (PNSP) recovered from children with acute otitis media (AOM) is unclear. METHODS: At 5 hospitals, 505 pneumococcal isolates were collected from children with AOM between 1 January 1999 and 31 December 2002. Molecular subtyping was performed on 158 isolates. RESULTS: Overall, the percentage of AOM cases due to non-PCV7 serogroups (including serotype 3) increased over time (from 12% in 1999 to 32% in 2002; P < .01) and according to the number of PCV7 doses received (18% [< or = 1 dose] vs. 35% [2-4 doses]; P < .01). The percentage of cases due to vaccine-related serotypes (including serotype 19A) increased according to the number of PCV7 doses received (10% [< or = 1 dose] vs. 19% [2-4 doses]; P = .05) but not over time, whereas the percentage of cases due to serotype 19F remained unchanged both over time and according to the number of PCV7 doses received. The frequency of penicillin nonsusceptibility among PCV7 serotypes (range, 65%-75%) and non-PCV7 serogroups (range, 11%-27%) did not significantly change overall. Although no change was detected among isolates collected from children with spontaneous drainage, the percentage of pneumococci recovered at the time of myringotomy and/or tympanostomy tube placement that were nonresistant to penicillin decreased over time (from 73% in 1999 to 53% in 2002; P = .03). All of the serotype 3 strains were genetically related, whereas 88% of the isolates that were either serotype 19F or serotype 23F were related to 1 of 3 international clones. CONCLUSIONS: Among children with AOM, the proportion of cases due to non-PCV7 serogroups increased, vaccine-related serotypes increased, and serotype 19F remained unchanged. Although a decrease in the proportion of cases due to PNSP occurred among children who required myringotomy and/or tympanostomy tube placement, the proportion of PNSP remained unchanged overall and among children with spontaneous drainage. Because future trends in the susceptibility patterns of pneumococcal isolates recovered from children with AOM are not easy to predict, continued surveillance is essential.

Both cAMP and cGMP are required for maximal ciliary beat stimulation in a cell-free model of bovine ciliary axonemes.

Previously, we have shown that the ATPase-dependent motion of cilia in bovine bronchial epithelial cells (BBEC) can be regulated through the cyclic nucleotides, cAMP via the cAMP-dependent protein kinase (PKA) and cGMP via the cGMP-dependent protein kinase (PKG). Both cyclic nucleotides cause an increase in cilia beat frequency (CBF). We hypothesized that cAMP and cGMP may act directly at the level of the ciliary axoneme in BBEC. To examine this, we employed a novel cell-free system utilizing detergent-extracted axonemes. Axoneme movement was whole-field analyzed digitally with the Sisson-Ammons video analysis system. A suspension of extracted axonemes remains motionless until the addition of 1 mM ATP that establishes a baseline CBF similar to that seen when analyzing intact ciliated BBEC. Adding 10 microM cAMP or 10 microM cGMP increases CBF beyond the established ATP baseline. However, the cyclic nucleotides did not stimulate CBF in the absence of ATP. Therefore, the combination of cAMP and cGMP augments ATP-driven CBF increases at the level of isolated axoneme.

Serotype 14 variants of the France 9V(-3) clone from Baltimore, Maryland, can be differentiated by the cpsB gene.

European serotype 14 variants of the France 9V(-3) clone, which have arisen through recombination events involving the penicillin binding protein 1a (pbp1a) gene, have cpsB sequences distinct from those of the 9V(-3) clone. Serotype 14 variants of the 9V(-3) clone have not been compared to genetically diverse serotype 14 strains isolated from an entire metropolitan area in the United States. All serotype 14 non-penicillin-susceptible Streptococcus pneumoniae strains causing invasive disease in Baltimore, Md., from 1995 to 1996 were compared by using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), pbp1a PCR restriction profiles, and cpsB and pbp1a sequences. The cpsB genes from strains of 13 serotypes also were analyzed to assess the correlation with serotype. Twenty-seven percent (3 of 11) of the serotype 14 strains were related by PFGE and MLST to the 9V(-3) clone. The serotype 14 variants from Baltimore, unlike the European variants, were related neither to the 9V(-3) clone nor to the R6 strain from positions 1498 to 1710 of the pbp1a gene. All serotype 14 strains had cpsB sequences that differed by or=16% (78 to 83 of 476 bp) divergent from that of the 9V(-3) clone. Allowing for a 2-bp difference in the cpsB sequence resulted in the highest correlation between the cpsB gene and serotype. Overall, 95% (84 of 88) of the strains were classified correctly by serotype with the cpsB sequence. The distal recombination site of the Baltimore serotype 14 variants of the 9V(-3) clone was not identical to that of the European serotype 14 variants. The cpsB gene was serotype specific regardless of whether capsular switching occurred. Although the correlation between serotype and the cpsB sequence was high, the overall diversity of the cpsB gene within a serotype likely will limit the role of this gene in a sequence-based serotyping method.