The objective of this study was to investigate the trends and patterns of resistance in β-lactamase-producing members of the familyEnterobacteriaceaein a children's hospital over a 9-year period (1999 to 2007). Clinically significant isolates of theEnterobacteriaceaewere screened for patterns of broad-spectrum resistance to β-lactams. The strains likely to be resistant were subsequently confirmed by an inhibitor-based disc test. The plasmid-mediated resistance determinants in these isolates were identified by PCR and by in vitro transformation, which successfully reproduced the AmpC phenotype unrestricted by the species of the host organisms. Among 8,048Enterobacteriaceaeisolates belonging to the four chromosomalampC-negative or -nonfunctional genera, 86 (1.07%) isolates (56Escherichia coliisolates, 22Klebsiellaspecies isolates, 1Proteus mirabilisisolate, and 7Salmonellaspecies isolates) exhibited broad-spectrum β-lactam resistance patterns. These organisms collectively produced three classes of β-lactamases, including class A extended-spectrum β-lactamases (n= 47), class C or AmpC β-lactamases (n= 36, including 4 isolates that produced both class A and class C enzymes), and class A or B carbapenem-hydrolyzing β-lactamases (n= 3). The proportion increased from 0.46% during the first 3 years to 1.84% during the last 3 years (relative risk [RR], 4.04; 95% confidence interval [CI], 2.28 to 7.42;P< 0.001). The increase was mainly due to the emergence of a plasmid-mediatedbla_CMY-2β-lactamase, the incidence of which increased from 0.11% during the first 3 years to 0.96% during the last 3 years (RR, 9.11; 95% CI, 2.76 to 30.1;P= 0.001). Class A-type resistance increased slightly during the study period, from 0.35% during the first 3 years to 0.85% during the last 3 years (RR, 2.42; 95% CI, 1.15 to 5.07;P= 0.02). AProteus mirabilisstrain was documented to possess a novelbla_DHAdeterminant. Of special concern, three carbapenemase-producing isolates were identified between 2003 and 2006. The infections caused by resistant isolates of theEnterobacteriaceaemainly affected hospitalized patients with underlying conditions; however, 19 (22%) episodes were of community onset in otherwise well children. The rate of resistance to broad-spectrum β-lactams among isolates of theEnterobacteriaceaeis increasing in children in both hospital- and community-acquired settings, and the resistance is driven largely by plasmid-mediated AmpC β-lactamases. These data have important implications for empirical antimicrobial strategies targeting serious pediatric infections. Further study of this problem is warranted.

Cystic fibrosis (CF) patients are predisposed to chronic respiratory infection by nonfermentative gram-negative bacilli, including Stenotrophomonas maltophilia. S. maltophilia is highly resistant to most antibiotics, with the exception of sulfamethoxazole-trimethoprim (SXT). SXT-resistant S. maltophilia has been reported, but the mechanism of resistance is not well defined. Repeated findings of suspected small-colony-variant (SCV) S. maltophilia isolates from the sputa of five CF patients were confirmed by partial 16S rRNA gene sequencing. The SCV S. maltophilia isolates were the only S. maltophilia isolates in these cultures, and none were clonally related. DNA fingerprint analysis confirmed that once established, the SCV S. maltophilia strains persisted. Nutritional studies of SCV S. maltophilia have suggested auxotrophy in hemin, methionine, and thymidine associated with resistance to multiple antibiotics, including SXT. The phenotypic switch from wild-type to SCV S. maltophilia was reproducible in vitro by exposure to SXT, suggesting that prolonged exposure to antibiotics may select for both the SCV S. maltophilia phenotype and SXT resistance by interference with the dihydrofolate reductase pathway. Recovery of SCV S. maltophilia from the sputum of CF patients has implications for both laboratory testing and patient management.

Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients undergo remarkable phenotypic divergence over time, including loss of pigmentation, hemolysis, motility, and quorum sensing and emergence of antibiotic hypersusceptibility and/or auxotrophism. With prolonged antibiotic treatment and steady decline in lung function in chronically infected patients, the divergent characteristics associated with CF isolates have traditionally been regarded as “adapted/unusual virulence,” despite the degenerative nature of these adaptations. We examined the phenotypic and genotypic diversity in clonally related isogenic strains of P. aeruginosa from individual CF patients. Our observations support a novel model of intra-airway pseudomonal syntrophy and accompanying loss of virulence. A 2007 calendar year collection of CF P. aeruginosa isolates ( n = 525) from 103 CF patients yielded in vitro MICs of sulfamethoxazole-trimethoprim (SMX-TMP, which typically has no activity against P. aeruginosa ) ranging from 0.02 to >32 μg/ml (median, 1.5). Coisolation of clonally related SMX-TMP-susceptible and -resistant P. aeruginosa strains from the same host was common (57%), as were isogenic coisolates with mutations in efflux gene determinants ( mexR , mexAB-oprM , and mexZ ) and genes governing DNA mismatch repair ( mutL and mutS ). In this cohort, complete in vitro growth complementation between auxotrophic and prototrophic P. aeruginosa isogenic strains was evident and concurrent with the coding sequence mosaicism in resistance determinants. These observations suggest that syntrophic clonal strains evolve in situ in an organized colonial structure. We propose that P. aeruginosa adopts a multicellular lifestyle in CF patients due to host selection of an energetically favorable, less-virulent microbe restricted within and symbiotic with the airway over the host's lifetime.

Timely accurate diagnosis of Shiga toxin-producing Escherichia coli (STEC) infections is important. We evaluated a laboratory-developed real-time PCR (LD-PCR) assay targeting stx ₁ , stx ₂ , and rfbE _O157 with 2,386 qualifying stool samples submitted to the microbiology laboratory of a tertiary care pediatric center between July 2011 and December 2013. Broth cultures of PCR-positive samples were tested for Shiga toxins by enzyme immunoassay (EIA) (ImmunoCard STAT! enterohemorrhagic E. coli [EHEC]; Meridian Bioscience) and cultured in attempts to recover both O157 and non-O157 STEC. E. coli O157 and non-O157 STEC were detected in 35 and 18 cases, respectively. Hemolytic uremic syndrome (HUS) occurred in 12 patients (10 infected with STEC O157, one infected with STEC O125ac, and one with PCR evidence of STEC but no resulting isolate). Among the 59 PCR-positive STEC specimens from 53 patients, only 29 (54.7%) of the associated specimens were toxin positive by EIA. LD-PCR differentiated STEC O157 from non-O157 using rfbE _O157 , and LD-PCR results prompted successful recovery of E. coli O157 ( n = 25) and non-O157 STEC ( n = 8) isolates, although the primary cultures and toxin assays were frequently negative. A rapid “mega”-multiplex PCR (FilmArray gastrointestinal panel; BioFire Diagnostics) was used retrospectively, and results correlated with LD-PCR findings in 25 (89%) of the 28 sorbitol-MacConkey agar culture-negative STEC cases. These findings demonstrate that PCR is more sensitive than EIA and/or culture and distinguishes between O157 and non-O157 STEC in clinical samples and that E. coli O157:H7 remains the predominant cause of HUS in our institution. PCR is highly recommended for rapid diagnosis of pediatric STEC infections.

PCR has greatly facilitated pertussis diagnosis due to the speed, sensitivity, and specificity of this assay compared to other detection methods. Various single-target PCR assays are currently utilized, but none is universally considered to be the “gold standard.” Our aim was to assess the use of multitarget versus single-target PCR for the diagnosis of pertussis in clinical samples. PCR assays targeting insertion sequence IS 481 (IS), pertussis toxin ptxA promoter region (PT), and outer membrane porin (PO), or recA (RA) were evaluated in respiratory specimens collected from 4,442 patients with suspected pertussis. The diagnosis of pertussis was confirmed in 309 (6.96%) patients by the 3-target IS-PT-PO/RA PCR versus 247 (5.56%) by the conventional single-target IS ( P = 0.007). Compared to single-target IS, the three-target combination increased the proportion of positive specimens by 1.25-fold, and two-target combinations increased the proportion of positive specimens by 1.10- to 1.24-fold. In addition, nine cases of B. parapertussis infection were also confirmed by using the discriminative features of this multitarget PCR. Of the 89 culture-proven pertussis cases, 17 (19.1%) and 5 of the 16 patients (31.3%) admitted to intensive care unit would have been missed had only the single-target IS PCR been applied. Patients with mild disease ( P = 0.004) and shorter hospitalization ( P = 0.006) were less likely to have positive cultures. This consensus generating real-time PCR approach permits a sensitive detection, as well as an accurate species identification of the causative Bordetella pathogens for the timely management of patients.

Staphylococcus aureus are Gram-positive bacteria that are the leading cause of recurrent infections in humans that include pneumonia, bacteremia, osteomyelitis, arthritis, endocarditis, and toxic shock syndrome. The emergence of methicillin resistant S. aureus strains (MRSA) has imposed a significant concern in sustained measures of treatment against these infections. Recently, MRSA strains deficient in expression of a serine/threonine kinase (Stk1 or PknB) were described to exhibit increased sensitivity to β-lactam antibiotics. In this study, we screened a library consisting of 280 drug-like, low-molecular-weight compounds with the ability to inhibit protein kinases for those that increased the sensitivity of wild-type MRSA to β-lactams and then evaluated their toxicity in mice. We report the identification of four kinase inhibitors, the sulfonamides ST085384, ST085404, ST085405, and ST085399 that increased sensitivity of WT MRSA to sub-lethal concentrations of β-lactams. Furthermore, these inhibitors lacked alerting structures commonly associated with toxic effects, and toxicity was not observed with ST085384 or ST085405 in vivo in a murine model. These results suggest that kinase inhibitors may be useful in therapeutic strategies against MRSA infections.