Dominant Carbapenemase-Encoding Plasmids in Clinical Enterobacterales Isolates and Hypervirulent Klebsiella pneumoniae, Singapore.

Dissemination of carbapenemase-encoding plasmids by horizontal gene transfer in multidrug-resistant bacteria is the major driver of rising carbapenem-resistance, but the conjugative mechanics and evolution of clinically relevant plasmids are not yet clear. We performed whole-genome sequencing on 1,215 clinical Enterobacterales isolates collected in Singapore during 2010-2015. We identified 1,126 carbapenemase-encoding plasmids and discovered pKPC2 is becoming the dominant plasmid in Singapore, overtaking an earlier dominant plasmid, pNDM1. pKPC2 frequently conjugates with many Enterobacterales species, including hypervirulent Klebsiella pneumoniae, and maintains stability in vitro without selection pressure and minimal adaptive sequence changes. Furthermore, capsule and decreasing taxonomic relatedness between donor and recipient pairs are greater conjugation barriers for pNDM1 than pKPC2. The low fitness costs pKPC2 exerts in Enterobacterales species indicate previously undetected carriage selection in other ecological settings. The ease of conjugation and stability of pKPC2 in hypervirulent K. pneumoniae could fuel spread into the community.

Cell envelope defects of different capsule-null mutants in K1 hypervirulent Klebsiella pneumoniae can affect bacterial pathogenesis.

Hypervirulent Klebsiella pneumoniae (hvKP) causes Klebsiella-induced liver abscess. Capsule is important for the pathogenesis of Klebsiella in systemic infection, but its role in gut colonisation is not well understood. By generating ΔwcaJ, Δwza and Δwzy capsule-null mutants in a prototypical K1 hypervirulent isolate, we show that inactivation of wza (capsule exportase) and wzy (capsule polymerase) confer cell envelope defects in addition to capsule loss, making them susceptible to bile salts and detergent stress. Bile salt resistance is restored when the initial glycosyltransferase wcaJ was inactivated together with wzy, indicating that build-up of capsule intermediates contribute to cell envelope defects. Mouse gut colonisation competition assays show that the capsule and its regulator RmpA were not required for hvKP to persist in the gut, although initial colonisation was decreased in the mutants. Both ΔrmpA and ΔwcaJ mutants gradually outcompeted the wild type in the gut, whereas Δwza and Δwzy mutants were less fit than wild type. Together, our results advise caution in using the right capsule-null mutant for determination of capsule's role in bacterial pathogenesis. With the use of ΔwcaJ mutant, we found that although the capsule is important for bacterial survival outside the gut environment, it imposes a fitness cost in the gut.

A Video-Based Classification System for Assessing Locomotor Skills in Children.

The Test of Gross Motor Development 2 (TGMD-2) is currently the standard approach for assessing fundamental movement skills (FMS), including locomotor and object control skills. However, its extensive application is restricted by its low efficiency and requirement of expert training for large-scale evaluations. This study evaluated the accuracy of a newly-developed video-based classification system (VCS) with a marker-less sensor to assess children's locomotor skills. A total of 203 typically-developing children aged three to eight years executed six locomotor skills, following the TGMD-2 guidelines. A Kinect v2 sensor was used to capture their activities, and videos were recorded for further evaluation by a trained rater. A series of computational-kinematic-based algorithms was developed for instant performance rating. The VCS exhibited moderate-to-very good levels of agreement with the rater, ranging from 66.1% to 87.5%, for each skill, and 72.4% for descriptive ratings. Paired t-test revealed that there were no significant differences, but significant positive correlation, between the standard scores determined by the two approaches. Tukey mean difference plot suggested there was no bias, with a mean difference (SD) of -0.16 (1.8) and respective 95% confidence interval of 3.5. The kappa agreement for the descriptive ratings between the two approaches was found to be moderate (k = 0.54, p < 0.01). Overall, the results suggest the VCS could potentially be an alternative to the conventional TGMD-2 assessment approach for assessing children's locomotor skills without the necessity of the presence of an experienced rater for the administration.

Hypervirulent Klebsiella pneumoniae employs genomic island encoded toxins against bacterial competitors in the gut.

The hypervirulent lineages of Klebsiella pneumoniae (HvKp) cause invasive infections such as Klebsiella-liver abscess. Invasive infection often occurs after initial colonization of the host gastrointestinal tract by HvKp. Over 80% of HvKp isolates belong to the clonal group 23 sublineage I that has acquired genomic islands (GIs) GIE492 and ICEKp10. Our analysis of 12 361 K. pneumoniae genomes revealed that GIs GIE492 and ICEKp10 are co-associated with the CG23-I and CG10118 HvKp lineages. GIE492 and ICEKp10 enable HvKp to make a functional bacteriocin microcin E492 (mccE492) and the genotoxin colibactin, respectively. We discovered that GIE492 and ICEKp10 play cooperative roles and enhance gastrointestinal colonization by HvKp. Colibactin is the primary driver of this effect, modifying gut microbiome diversity. Our in vitro assays demonstrate that colibactin and mccE492 kill or inhibit a range of Gram-negative Klebsiella species and Escherichia coli strains, including Gram-positive bacteria, sometimes cooperatively. Moreover, mccE492 and colibactin kill human anaerobic gut commensals that are similar to the taxa found altered by colibactin in the mouse intestines. Our findings suggest that GIs GIE492 and ICEKp10 enable HvKp to kill several commensal bacterial taxa during interspecies interactions in the gut. Thus, acquisition of GIE492 and ICEKp10 could enable better carriage in host populations and explain the dominance of the CG23-I HvKp lineage.

Acquisition of regulator on virulence plasmid of hypervirulent Klebsiella allows bacterial lifestyle switch in response to iron.

Hypervirulent Klebsiella pneumoniae causes liver abscess and potentially devastating metastatic complications. The majority of Klebsiella-induced liver abscess are caused by the CG23-I sublineage of hypervirulent Klebsiella pneumoniae. This and some other lineages possess a >200-kb virulence plasmid. We discovered a novel protein IroP nestled in the virulence plasmid-encoded salmochelin operon that cross-regulates and suppresses the promoter activity of chromosomal type 3 fimbriae (T3F) gene transcription. IroP is itself repressed by iron through the ferric uptake regulator. Iron-rich conditions increase T3F and suppress capsule mucoviscosity, leading to biofilm formation and cell adhesion. Conversely, iron-poor conditions cause a transcriptional switch to hypermucoid capsule production and T3F repression. The likely acquisition of iroP on mobile genetic elements and successful adaptive integration into the genetic circuitry of a major lineage of hypervirulent K. pneumoniae reveal a powerful example of plasmid chromosomal cross talk that confers an evolutionary advantage. Our discovery also addresses the conundrum of how the hypermucoid capsule that impedes adhesion could be regulated to facilitate biofilm formation and colonization. The acquired ability of the bacteria to alternate between a state favoring dissemination and one that favors colonization in response to iron availability through transcriptional regulation offers novel insights into the evolutionary success of this pathogen. IMPORTANCE Hypervirulent Klebsiella pneumoniae contributes to the majority of monomicrobial-induced liver abscess infections that can lead to several other metastatic complications. The large virulence plasmid is highly stable in major lineages, suggesting that it provides survival benefits. We discovered a protein IroP encoded on the virulence plasmid that suppresses expression of the type 3 fimbriae. IroP itself is regulated by iron, and we showed that iron regulates hypermucoid capsule production while inversely regulating type 3 fimbriae expression through IroP. The acquisition and integration of this inverse transcriptional switch between fimbriae and capsule mucoviscosity shows an evolved sophisticated plasmid-chromosomal cross talk that changes the behavior of hypervirulent K. pneumoniae in response to a key nutrient that could contribute to the evolutionary success of this pathogen.

The cefoperazone-sulbactam combination. In vitro qualities including beta-lactamase stability, antimicrobial activity, and interpretive criteria for disk diffusion tests.

Three concentrations of the penicillanic acid sulfone, sulbactam were tested in combination with cefoperazone against 632 recent clinical bacterial isolates. Cefoperazone was effective alone (less than or equal to 16 micrograms/mL) against 95% of Enterobacteriaceae and combined with 4 micrograms/mL sulbactam inhibited 99.5% of strains. This coverage of enteric bacilli was superior to timentin (99.1%), ceftazidime (98.2%), and tobramycin (90.9%). The minimum inhibitory concentrations (MICs) of cefoperazone-susceptible strains also were markedly decreased by sulbactam (overall MIC90s, 8.0 micrograms/mL for cefoperazone and 1.0 microgram/mL for cefoperazone and 4.0 micrograms/mL for sulbactam). Sulbactam also expanded the spectrum of cefoperazone against Acinetobacter species, some rare Pseudomonas species, and Bacteroides fragilis group species. Sulbactam had direct antimicrobial activity against the acinetobacters and Pseudomonas acidovorans, but the increased activity of cefoperazone-sulbactam against some other Pseudomonas species and anaerobes was attributed to beta-lactamase inhibition. The cefoperazone MICs against beta-lactamase producing Staphylococcus species also were lowered to the level of enzyme-deficient strains. Cefoperazone bactericidal activity was improved by 4.0 micrograms/mL sulbactam, and no antagonism was observed. beta-lactamase hydrolysis studies confirmed a slow hydrolysis of cefoperazone only by TEM beta-lactamases and a high-grade resistance to enzyme breakdown by sulbactam. Differential beta-lactamase affinity studies for cefoperazone and sulbactam showed potential efficacy and applications to plasmid-mediated TEM and OXA enzymes and only marginal effective sulbactam inhibition of Pseudomonas and Klebsiella species enzymes. Disk diffusion studies on 556 strains confirmed the applicability of the cefoperazone 75-micrograms disk to testing routine isolates other than enterococci and methicillin-resistant Staphylococcus aureus. The addition of 4.0 micrograms sulbactam/mL in a fixed concentration to dilution test systems and 15 micrograms sulbactam to the 75 micrograms cefoperazone disk were recommended for in vitro tests. Susceptibility and resistant interpretive criteria for the disk and dilution tests can be applied with confidence. Only 0.4% false-susceptibility errors and a 97.5% absolute interpretive agreement were achieved using the 75 micrograms cefoperazone/15 micrograms sulbactam disk.

Antimicrobial activity, beta-lactamase stability and beta-lactamase inhibition of cefotetan and other 7-alpha-methoxy beta-lactam antimicrobials.

The antimicrobial activity of three 7-alpha-methoxy beta-lactams were compared to cefoperazone and ceftriaxone. All had a similar spectrum of activity against the Enterobacteriaceae, except cefoxitin. Cefotetan was only slightly less active than moxalactam against the Enterobacter spp. Ceftriaxone was most effective on Neisserias, Haemophilus spp, nonenterococcal Streptococcus spp, and Acinetobacter spp. Cefoperazone generally inhibited more pseudomonads while all of the "cephamycins" showed activity against Bacteroides fragilis and B. thetaiotaomicron. Beta-lactamase hydrolysis studies of six substrates having pharmacologic serum half lives of greater than or equal to 2 hrs were performed by bioassay and automated procedures. Excellent correlations were found between methods up to 24 hrs. A "lag-phase" was observed for several drug/enzyme combinations before initiation of significant substrate hydrolysis. The 7-alpha-methoxy beta-lactams were routinely more stable to the six representative enzymes (Richmond-Sykes types I-V) than other "stable" cephalosporins. Substrate hydrolysis rates resulting in greater than 50% drug loss in less than or equal to 1 hr generally produced resistant in vitro test results. Cefotetan, cefoxitin, moxalactam, ceftriaxone, and dicloxacillin were potent inhibitors of Type I (P99) beta-lactamases. Moxalactam demonstrated significant inhibition and affinity for the Type V enzyme while cefoperazone uniquely possesses affinity (so-called inhibition) for all tested beta-lactamases. Cefotetan appears to be a promising, beta-lactam compound with some in vitro characteristics comparable to the 1-oxa-beta-lactams and alpha-methoxyimino cephalosporins.

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.

In vitro antimicrobial activity of cefoperazone-sulbactam combinations against 554 clinical isolates including a review and beta-lactamase studies.

Cefoperazone was tested against 554 clinical isolates alone and with sulbactam in three combinations. The addition of sulbactam in low concentrations (less than or equal to 4 micrograms/ml) improved the spectrum of cefoperazone principally against gram-negative bacilli such as Acinetobacter species, some Pseudomonas species, and beta-lactamase-positive Enterobacteriaceae. Nearly all of the spectrum increase was achieved at a sulbactam level of less than or equal to 2 micrograms/ml. Sulbactam was found to be an effective antimicrobial agent against Acinetobacter species (MIC50, 1.0 microgram/ml), Pseudomonas acidovorans (MIC50, 2.0 micrograms/ml), Neisseria gonorrhoeae (MIC50, less than or equal to 0.5 microgram/ml), and N. meningitidis (MIC50, less than or equal to 0.5 microgram/ml). Sulbactam had a higher affinity and binding constant for the plasmid-mediated beta-lactamases such as TEM-1 and TEM-2 compared to cefoperazone (greater than or equal to 10-fold difference). This finding was important as cefoperazone can be hydrolyzed at a moderate rate by the highly efficient TEM enzymes (less than 2% of clinical Escherichia coli isolates). Sulbactam increased the susceptibility (less than or equal to 16 micrograms/ml) of 220 isolates of Enterobacteriaceae to cefoperazone from 88.6 to 96.3% when 4.0 micrograms/ml of sulbactam was added. The cefoperazone antimicrobial activity was also increased against the nonenteric bacilli from a 69.5 to a 87.4% total inhibition. MICs among cefoperazone-susceptible gram-negative and gram-positive strains were routinely decreased 2- to 32-fold, as calculated from MIC90 results. Therefore, sulbactam should predictably increase the antimicrobial spectrum and clinical effectiveness of cefoperazone against nosocomial and other pathogens such as the plasmid-containing enteric bacilli, Bacteroides species and Acinetobacter species, and possibly provide the opportunity to reduce dosage schedules for infecting species already susceptible to cefoperazone alone.

Infrared absorption bands of alpha and beta -pinenes in the 8-14-microm atmospheric window region.

There are bands in the 8 -14-microm atmospheric window region of the gas phase IR spectra of alpha- and beta-pinenes that are intense enough to allow theoretically the naturally occurring terpenes to be determined in the 100- ppbv level at 1-km path lengths. Because of the broadness of the bands, however, and the relatively intense atmospheric background at their frequencies of 786.5 cm(-1) and 880 cm(-1), respectively, practical concentration limits for the use of the bands are probably not better than about 1-10 ppmv. Both type C bands have 4-cm(-1)Q branches which fall at frequencies which are essentially free of any major atmospheric fine structure bands.

Comparative beta-lactamase hydrolysis of and inhibition by 7-aminothiazolyl alpha-methoxyimino cephalosporins.

Six 7-aminothiazolyl alpha-methoxyimino cephalosporins were found to be most variable in their ability to resist Type IV beta-lactamases (ceftizoxime, most stable and ceftriaxone, least stable), to inhibit Type I beta-lactamases (cefotaxime, the best inhibitor and desacetyl-cefotaxime, the least inhibition), to inhibit Enterobacteriaceae as measured by their minimum inhibitory concentrations (MICs), and in their published serum half-lives. The 3-position substituents appear to exert significant physical-chemical effects that influence the pharmacology, molecular enzyme stability and the antimicrobial activity of these compounds. However, we conclude that these differences are of minimal clinical consequence and that other more relevant factors for in vivo drug selection should be considered, including proven clinical efficacy and cost.

In vitro evaluation of pyridine-2-azo-p-dimethylaniline cephalosporin, a new diagnostic chromogenic reagent, and comparison with nitrocefin, cephacetrile, and other beta-lactam compounds.

Pyridine-2-azo-p-dimethylanaline cephalosporin (PADAC), a chromogenic reagent which is purple and changes to yellow upon cleavage of its beta-lactam ring, was evaluated in comparison with other chromogenic cephalosporins. PADAC exhibited little antimicrobial activity against gram-negative bacteria, but did have good activity (minimum inhibitory concentration, 0.12 to 0.5 microgram/ml) against Staphylococcus aureus, a quality comparable to nitrocefin. Nitrocefin, however, demonstrated an unexpected and uniquely potent activity against Streptococcus faecalis (minimum inhibitory concentration, less than or equal to 0.06 to 0.12 microgram/ml) The relative hydrolysis rate of PADAC when subjected to six different beta-lactamases was substantially greater than that of cephacetrile, but less than that of nitrocefin. The relative hydrolysis rates of PADAC and nitrocefin were comparable with type IIIa beta lactamase and the derived from Bacillus cereus. The inhibition of beta-lactamase hydrolysis of the chromogenic cephalosporin substrates by six enzyme-stable inhibitors was generally greater with PADAC than with nitrocefin. Unlike nitrocefin, PADAC mixed with 50% human serum or various broth culture media showed no evidence of color change or degradation over several hours. The subsequent enzyme hydrolysis rates of such mixtures were the same as in phosphate buffer. Beta-lactamase-containing bacterial suspensions and clinical specimens containing such bacteria produced positive visual and spectrophotometric color changes when mixed with PADAC or nitrocefin. Although color changes occurred more slowly with PADAC than with nitrocefin, PADAC was not adversely influenced (non-enzyme-related color change) by the protein content of specimens. PADAC appears to be a promising alternative for beta-lactamase diagnostic testing in the clinical and research microbiology laboratory.

In vitro antimicrobial activity evaluation of cefodizime (HR221), a new semisynthetic cephalosporin.

Cefodizime (HR221) is a new alpha-methoxyimino cephalosporin developed by Hoechst-Roussel with a reported serum half-life of over 2 h. In vitro susceptibility studies showed that the cefodizime spectrum includes all those Enterobacteriaceae, staphylococci, Streptococcus spp., Haemophilus spp., and Neisseria spp. normally susceptible to cefotaxime (HR756) or ceftizoxime (FK749) or both. Cefodizime was less active (two- to eightfold) than cefotaxime or ceftizoxime against some enteric species, but was the most potent drug against some strains of Morganella spp. and Proteus vulgaris. Enterococci, methicillin-resistant Staphylococcus aureus, and most Pseudomonas spp. were resistant to cefodizime (median minimum inhibitory concentrations, greater than or equal to 64 microgram/ml). Acinetobacter spp. and Pseudomonas aeruginosa strains required cefodizime concentrations of 32 microgram/ml to inhibit 50% of tested strains. Cefodizime was very stable to hydrolysis by Richmond-Sykes type I, II, III, and IV beta-lactamases, as well as the enzyme derived from Bacillus cereus. The reference PADAC and nitrocefin substrate hydrolysis by a type I beta-lactamase was markedly inhibited (greater than 80%) by cefodizime at concentrations 0.4 to 4% of substrate concentration. Cefodizime was active against 43% of bacteria which were resistant to cephalothin and cefamandole and against 58% of those resistant to aminoglycosides.

In vitro evaluation of CENTA, a new beta-lactamase-susceptible chromogenic cephalosporin reagent.

CENTA is a newly synthesized, beta-lactamase-labile, chromogenic cephalosporin reagent which changes color from light yellow (lambda maximum ca. 340 nm) to chrome yellow (lambda maximum ca. 405 nm) concomitant with hydrolysis of the beta-lactam ring. This compound offers promise as a diagnostic reagent comparable to other chromogens (PADAC and nitrocefin) for the early detection of beta-lactamase-producing clinical isolates, while retaining some antimicrobial effect against Escherichia coli, Klebsiella spp., Proteus mirabilis, Staphylococcus aureus, and non-enterococcal Streptococcus spp. CENTA is relatively unaffected by commonly used microbiological media and human serum.