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Klebsiella oxytoca complex comprises nine closely related species causing human infections. We curated genomes labeled Klebsiella (n = 14,256) in GenBank and identified 588 belonging to the complex, which were examined for precise species, sequence types, K- and O-antigen types, and virulence and antimicrobial resistance genes. The complex and Klebsiella pneumoniae share many K- and O-antigen types. Of the complex, K. oxytoca and Klebsiella michiganensis appear to carry more virulence genes and be more commonly associated with human infections.

Third-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae represent a major threat to human health. Here, we captured 288 3GC-R Enterobacteriaceae clinical isolates from 264 patients presenting at a regional Australian hospital over a 14-month period. In addition to routine mass spectrometry and antibiotic sensitivity testing, isolates were examined using rapid (∼40-min) real-time PCR assays targeting the most common extended-spectrum β-lactamases (ESBLs; bla(CTX-M-1) and bla(CTX-M-9) groups, plus bla(TEM), bla(SHV), and an internal 16S rRNA gene control). AmpC CMY β-lactamase (bla(CMY)) prevalence was also examined. Escherichia coli (80.2%) and Klebsiella pneumoniae (17.0%) were dominant, with Klebsiella oxytoca, Klebsiella aerogenes, and Enterobacter cloacae infrequently identified. Ceftriaxone and cefoxitin resistance were identified in 97.0% and 24.5% of E. coli and K. pneumoniae isolates, respectively. Consistent with global findings in Enterobacteriaceae, most (98.3%) isolates harbored at least one β-lactamase gene, with 144 (50%) harboring bla(CTX-M-1) group, 92 (31.9%) harboring bla(CTX-M-9) group, 48 (16.7%) harboring bla(SHV), 133 (46.2%) harboring bla(TEM), and 34 (11.8%) harboring bla(CMY) genes. A subset of isolates (n = 98) were subjected to whole-genome sequencing (WGS) to identify the presence of cryptic resistance determinants and to verify genotyping accuracy. WGS of β-lactamase-negative or carbapenem-resistant isolates identified uncommon ESBL and carbapenemase genes, including bla(NDM) and bla(IMP), and confirmed all PCR-positive genotypes. We demonstrate that our PCR assays enable the rapid and cost-effective identification of ESBLs in the hospital setting, which has important infection control and therapeutic implications.

In this study, we investigated VIM-1-producing Escherichia coli , Klebsiella oxytoca , Klebsiella pneumoniae , Citrobacter freundii , and Enterobacter cloacae strains, isolated in 2019 during a period of active surveillance of carbapenem-resistant Enterobacterales in a large university hospital in Italy. VIM-1-producing strains colonized the gut of patients, with up to three different VIM-1-positive bacterial species isolated from a single rectal swab, but also caused bloodstream infection in one colonized patient.

Omadacycline is a broad-spectrum aminomethylcycline approved in October 2018 by the U.S. Food and Drug Administration for treating acute bacterial skin and skin structure infections and community-acquired pneumonia as both an oral and intravenous once-daily formulation. In this report, the activities of omadacycline and comparators were tested against 49,000 nonduplicate bacterial isolates collected prospectively during 2016 to 2018 from medical centers in Europe (24,500 isolates, 40 medical centers [19 countries]) and the United States (24,500 isolates, 33 medical centers [23 states and all 9 U.S. census divisions]). Omadacycline was tested by broth microdilution following the methods in Clinical and Laboratory Standards Institute document M07 (Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard, 11th ed., 2018). Omadacycline (MIC(50/90), 0.12/0.25 mg/liter) inhibited 98.6% of Staphylococcus aureus isolates at ≤0.5 mg/liter, including 96.3% of methicillin-resistant S. aureus isolates and 99.8% of methicillin-susceptible S. aureus isolates. Omadacycline potency was comparable for Streptococcus pneumoniae (MIC(50/90), 0.06/0.12 mg/liter), viridans group streptococci (MIC(50/90), 0.06/0.12 mg/liter), and beta-hemolytic streptococci (MIC(50/90), 0.12/0.25 mg/liter), regardless of species and susceptibility to penicillin, macrolides, or tetracycline. Omadacycline was active against all Enterobacterales tested (MIC(50/90), 1/8 mg/liter; 87.5% of isolates were inhibited at ≤4 mg/liter) except Proteus mirabilis (MIC(50/90), 16/>32 mg/liter) and indole-positive Proteus spp. (MIC(50/90), 8/32 mg/liter) and was most active against Escherichia coli (MIC(50/90), 0.5/2 mg/liter), Klebsiella oxytoca (MIC(50/90), 1/2 mg/liter), and Citrobacter spp. (MIC(50/90), 1/4 mg/liter). Omadacycline inhibited 92.4% of Enterobacter cloacae species complex and 88.5% of Klebsiella pneumoniae isolates at ≤4 mg/liter. Omadacycline was active against Haemophilus influenzae (MIC(50/90), 0.5/1 mg/liter), regardless of β-lactamase status, and against Moraxella catarrhalis (MIC(50/90), ≤0.12/0.25 mg/liter). The potent activity of omadacycline against Gram-positive and -negative bacteria indicates that omadacycline merits further study in serious infections in which multidrug resistance and mixed Gram-positive and Gram-negative bacterial infections may be a concern.

The expression of the bla(KPC) gene plays a key role in carbapenem resistance in Enterobacteriaceae. However, the genetic regulators of the bla(KPC) gene have not been completely elucidated, especially the genes in Tn3-Tn4401 chimeras. Two novel Tn3-Tn4401 chimera isoforms were characterized in our hospital, isoform A (CTA), which harbors a 121-bp deletion containing the PX promoter and was present in 22.6% (54/239) of isolates, and isoform C (CTC), which harbors a 624-bp insertion and a P1 promoter deletion and was present in only 1 isolate. The carbapenem MICs of both isoforms were 2-fold or more higher than those of the wild type (Tn3-Tn4401 chimera, CTB), and bla(KPC) was most highly expressed in CTA. Bioinformatics and 5′ rapid amplification of cDNA ends (5′ RACE) experiments indicated a novel strong putative promoter, PY, at the 3′ end of the ISKpn8 gene. PY mutation nearly abrogated bla(KPC) expression (P

The recent development of new antimicrobials active against carbapenemase-producing Enterobacteriales (CPE) has brought new hope for the treatment of infections due to these organisms. However, the evolving epidemiology of bacteria with carbapenemases may complicate management, as providers are faced with treating patients colonized by bacteria producing multiple carbapenemases. Here, we present the clinical course and treatment of Raoultella planticola bacteremia in a cirrhotic patient known to be colonized with both bla(KPC)- and bla(OXA-48)-carrying organisms.

The in vitro activity of ceftazidime-avibactam was evaluated against 34,062 isolates of Enterobacteriaceae from patients with intra-abdominal, urinary tract, skin and soft-tissue, lower respiratory tract, and blood infections collected in the INFORM (International Network For Optimal Resistance Monitoring) global surveillance study (176 medical center laboratories in 39 countries) in 2012 to 2014. Overall, 99.5% of Enterobacteriaceae isolates were susceptible to ceftazidime-avibactam using FDA approved breakpoints (susceptible MIC of ≤8 μg/ml; resistant MIC of ≥16 μg/ml). For individual species of the Enterobacteriaceae , the ceftazidime-avibactam MIC inhibiting ≥90% of isolates (MIC 90 ) ranged from 0.06 μg/ml for Proteus species to 1 μg/ml for Enterobacter spp. and Klebsiella pneumoniae . Carbapenem-susceptible isolates of Escherichia coli , K. pneumoniae , Klebsiella oxytoca , and Proteus mirabilis with a confirmed extended-spectrum β-lactamase (ESBL) phenotype, or a ceftazidime MIC of ≥16 μg/ml if the ESBL phenotype was not confirmed by clavulanic acid inhibition, were characterized further to identify the presence of specific ESBL- and plasmid-mediated AmpC β-lactamase genes using a microarray-based assay and additional PCR assays. Ceftazidime-avibactam demonstrated potent activity against molecularly confirmed ESBL-producing ( n = 5,354; MIC 90 , 0.5 μg/ml; 99.9% susceptible), plasmid-mediated AmpC-producing ( n = 246; MIC 90 , 0.5 μg/ml; 100% susceptible), and ESBL- and AmpC-producing ( n = 152; MIC 90 , 1 μg/ml; 100% susceptible) isolates of E. coli , K. pneumoniae , K. oxytoca , and P. mirabilis . We conclude that ceftazidime-avibactam demonstrates potent in vitro activity against globally collected clinical isolates of Enterobacteriaceae , including isolates producing ESBLs and AmpC β-lactamases.

The imipenem-relebactam combination is in development as a potential treatment regimen for infections caused by Enterobacteriaceae possessing complex β-lactamase backgrounds. Relebactam is a β-lactamase inhibitor that possesses the diazabicyclooctane core, as in avibactam; however, the R1 side chain of relebactam also includes a piperidine ring, whereas that of avibactam is a carboxyamide. Here, we investigated the inactivation of the Klebsiella pneumoniae carbapenemase KPC-2, the most widespread class A carbapenemase, by relebactam and performed susceptibility testing with imipenem-relebactam using KPC-producing clinical isolates of Enterobacteriaceae . MIC measurements using agar dilution methods revealed that all 101 clinical isolates of KPC-producing Enterobacteriaceae ( K. pneumoniae , Klebsiella oxytoca , Enterobacter cloacae , Enterobacter aerogenes , Citrobacter freundii , Citrobacter koseri , and Escherichia coli ) were highly susceptible to imipenem-relebactam (MICs ≤ 2 mg/liter). Relebactam inhibited KPC-2 with a second-order onset of acylation rate constant ( k 2 / K ) value of 24,750 M −1 s −1 and demonstrated a slow off-rate constant ( k off ) of 0.0002 s −1 . Biochemical analysis using time-based mass spectrometry to map intermediates revealed that the KPC-2–relebactam acyl-enzyme complex was stable for up to 24 h. Importantly, desulfation of relebactam was not observed using mass spectrometry. Desulfation and subsequent deacylation have been observed during the reaction of KPC-2 with avibactam. Upon molecular dynamics simulations of relebactam in the KPC-2 active site, we found that the positioning of active-site water molecules is less favorable for desulfation in the KPC-2 active site than it is in the KPC-2–avibactam complex. In the acyl complexes, the water molecules are within 2.5 to 3 Å of the avibactam sulfate; however, they are more than 5 to 6 Å from the relebactam sulfate. As a result, we propose that the KPC-2–relebactam acyl complex is more stable than the KPC-2–avibactam complex. The clinical implications of this difference are not currently known.

Ceftazidime-avibactam (MIC 50/90 , 0.12/0.25 μg/ml) inhibited 99.9% (20,698/20,709) of Enterobacteriaceae isolates at ≤8 μg/ml. This compound was active against resistant subsets, including ceftazidime-nonsusceptible Enterobacter cloacae (MIC 50/90 , 0.25/0.5 μg/ml) and extended-spectrum β-lactamase (ESBL) phenotype isolates. An ESBL phenotype was noted among 12.4% (1,696/13,692 isolates from targeted species) of the isolates, including 776 Escherichia coli (12.0% for this species; MIC 50/90 , 0.12/0.25 μg/ml), 721 Klebsiella pneumoniae (16.3%; MIC 50/90 , 0.12/0.25 μg/ml), 119 Klebsiella oxytoca (10.3%; MIC 50/90 , 0.06/0.25 μg/ml), and 80 Proteus mirabilis (4.9%; MIC 50/90 , 0.06/0.12 μg/ml) isolates. The most common enzymes detected among ESBL phenotype isolates from 2013 ( n = 743) screened using a microarray-based assay were CTX-M-15-like ( n = 307), KPC ( n = 120), SHV ESBLs ( n = 118), and CTX-M-14-like ( n = 110). KPC producers were highly resistant to comparators, and ceftazidime-avibactam (MIC 50/90 , 0.5/2 μg/ml) and tigecycline (MIC 50/90 , 0.5/1 μg/ml; 98.3% susceptible) were the most active agents against these strains. Meropenem (MIC 50/90 , ≤0.06/≤0.06 μg/ml) and ceftazidime-avibactam (MIC 50/90 , 0.12/0.25 μg/ml) were active against CTX-M-producing isolates. Other enzymes were also observed, and ceftazidime-avibactam displayed good activity against the isolates producing less common enzymes. Among 11 isolates displaying ceftazidime-avibactam MIC values of >8 μg/ml, three were K. pneumoniae strains producing metallo-β-lactamases (all ceftazidime-avibactam MICs, >32 μg/ml), with two NDM-1 producers and one K. pneumoniae strain carrying the bla KPC-2 and bla VIM-4 genes. Therapeutic options for isolates producing β-lactamases may be limited, and ceftazidime-avibactam, which displayed good activity against strains, including those producing KPC enzymes, merits further study in infections where such organisms occur.

The nucleotide sequences of five plasmids from one Klebsiella oxytoca isolate were determined using the PacBio RS II system. Plasmid analysis revealed that bla NDM-1 was carried on an IncX3 plasmid. The bla IMP-4 and bla KPC-2 genes were located on IncN and IncP-6 plasmids, respectively. Comparative sequence analysis highlighted the successful spread of carbapenemase-harboring plasmids among different enterobacterial species. We report for the first time, to our knowledge, coproducing NDM-1, KPC-2, and IMP-4 carbapenemases on a K. oxytoca isolate.

Omadacycline is a broad-spectrum aminomethylcycline in late-stage clinical development for the treatment of acute bacterial skin and skin structure infections and community-acquired pneumonia as an oral and an intravenous once-daily formulation. In this study, omadacycline and comparators were tested against 69,246 nonduplicate bacterial isolates collected prospectively during 2010 and 2011 from medical centers in Asia-Pacific (11,397 isolates), Europe (23,490 isolates), Latin America (8,038 isolates), and North America (26,321 isolates). Omadacycline was tested by broth microdilution following Clinical and Laboratory Standards Institute M07-A10 (2015) methods. A total of 99.9% of Staphylococcus aureus isolates were inhibited by ≤2 μg/ml of omadacycline (MIC 50/90 , 0.12/0.25 μg/ml), including 100.0% of methicillin-susceptible S. aureus isolates and 99.8% of methicillin-resistant S. aureus isolates. Omadacycline potencies were comparable for Streptococcus pneumoniae (MIC 50/90 , 0.06/0.06 μg/ml), viridans group streptococci (MIC 50/90 , 0.06/0.12 μg/ml), and beta-hemolytic streptococci (MIC 50/90 , 0.06/0.12 μg/ml) regardless of species and susceptibility to penicillin. Omadacycline was active against Enterobacteriaceae and was most active against Escherichia coli (MIC 50/90 , 0.5/2 μg/ml), Enterobacter aerogenes (MIC 50/90 , 2/4 μg/ml), Klebsiella oxytoca (MIC 50/90 , 1/4 μg/ml), and Citrobacter spp. (MIC 50/90 , 1/4 μg/ml). Omadacycline was active against Haemophilus influenzae (MIC 50/90 , 1/1 μg/ml) regardless of β-lactamase status and against Moraxella catarrhalis (MIC 50/90 , 0.12/0.25 μg/ml). The potent activity of omadacycline against Gram-positive and Gram-negative bacteria indicates that omadacycline merits further study in serious infections in which multidrug resistance and mixed Gram-positive and Gram-negative infections may be a concern.

The Carba NP test was evaluated against a panel of 61 carbapenemase-producing bacterial species (15 producing class A carbapenemases, 15 producing class D carbapenemases, and 31 producing metallo-β-lactamases) and against 111 isolates with non-wild-type carbapenem susceptibility but not producing carbapenemase. Carbapenemase production was verified by PCR and UV-spectrophotometric measurement of imipenem hydrolysis. No false positives were seen, but there were consistent problems with the detection of OXA-48-like enzymes and also some rarer class A enzymes.

The increasing trend of β-lactam resistance among Enterobacteriaceae is a worldwide threat. Enterobacteriaceae isolates causing intra-abdominal infections (IAI) from the Study for Monitoring Antimicrobial Resistance Trends (SMART) collected in 2008 and 2009 from the Asia-Pacific region were investigated. Detection of extended-spectrum β-lactamases (ESBLs), AmpC β-lactamases, and carbapenemases was performed by multiplex PCR. A total of 699 Enterobacteriaceae isolates with positive genotypic results, included Escherichia coli ( n = 443), Klebsiella pneumoniae ( n = 187), Enterobacter cloacae ( n = 45), Klebsiella oxytoca ( n = 9), Citrobacter freundii ( n = 5), Proteus mirabilis ( n = 3), Enterobacter aerogenes ( n = 2), Morganella morganii ( n = 2), and one each of Enterobacter asburiae , Proteus vulgaris , and Providencia rettgeri were analyzed. Nearly 20% of these β-lactamase-producing Enterobacteriaceae isolates were from community-associated IAI. CTX-M (588 isolates, including 428 [72.8%] with CTX-M-15) was the most common ESBL, followed by SHV ( n = 59) and TEM ( n = 4). CMY ( n = 110, including 102 [92.7%] with CMY-2) was the most common AmpC β-lactamase, followed by DHA ( n = 46) and ACT/MIR ( n = 40). NDM ( n = 65, including 62 [95.4%] with NDM-1) was the most common carbapenemase, followed by IMP ( n = 7) and OXA ( n = 7). Isolates from hospital-associated IAI had more complicated β-lactamase combinations than isolates from the community. Carbapenemases were all exclusively detected in Enterobacteriaceae isolates from India, except that IMP β-lactamases were also detected in Philippines and Australia. CTX-M β-lactamases were the predominant ESBLs produced by Enterobacteriaceae causing IAI in the Asia-Pacific region. Emergence of CTX-M-15-, CMY-2-, and NDM-1-producing Enterobacteriaceae isolates is of major concern and highlights the need for further surveillance in this area.

Ceftazidime-avibactam (CAZ-AVI) is a recently approved β-lactam–β-lactamase inhibitor combination with the potential to treat serious infections caused by carbapenem-resistant organisms. Few patients with such infections were included in the CAZ-AVI clinical trials, and clinical experience is lacking. We present a case series of patients with infections caused by carbapenem-resistant Enterobacteriaceae (CRE) or Pseudomonas aeruginosa (CRPa) who were treated with CAZ-AVI salvage therapy on a compassionate-use basis. Physicians who had prescribed CAZ-AVI completed a case report form. We used descriptive statistics to summarize patient characteristics and treatment outcomes. We used the Wilcoxon rank sum test and Fisher's exact test to compare patients by treatment outcome. The sample included 36 patients infected with CRE and two with CRPa. The most common infections were intra-abdominal. Physicians categorized 60.5% of patients as having life-threatening infections. All but two patients received other antibiotics before CAZ-AVI, for a median of 13 days. The median duration of CAZ-AVI treatment was 16 days. Twenty-five patients (65.8%) concurrently received other antibiotics to which their pathogen was nonresistant in vitro . Twenty-eight patients (73.7%, 95% confidence interval [CI], 56.9 to 86.6%) experienced clinical and/or microbiological cure. Five patients (20.8%) with documented microbiological cure died, whereas 10 patients (71.4%) with no documented microbiological cure died ( P = 0.01). In three-quarters of cases, CAZ-AVI (alone or combined with other antibiotics) cured infections caused by carbapenem-resistant organisms, 95% of which had failed previous therapy. Microbiological cure was associated with improved survival. CAZ-AVI shows promising clinical results for infections for which treatment options are limited.

From June to September 2011, a total of 305 ertapenem-nonsusceptible Enterobacteriaceae isolates (MICs of ertapenem ≥ 1 μg/ml) were collected from 11 hospitals in different parts of Taiwan. The MICs of 12 antimicrobial agents against these isolates were determined using the broth microdilution method, and genes for carbapenemases were detected using PCR. Genotypes of isolates possessing carbapenemase genes were identified by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. The ertapenem-nonsusceptible Enterobacteriaceae isolates included Klebsiella pneumoniae ( n = 219), Escherichia coli ( n = 64), Enterobacter cloacae ( n = 15), and other species ( n = 7). Seven (2.3%) of the ertapenem-nonsusceptible Enterobacteriaceae isolates exhibited colistin MICs of >4 μg/ml, and 24 (7.9%) were not susceptible to tigecycline (MICs > 2 μg/ml). A total of 29 (9.5%) isolates carried genes encoding carbapenemases, namely, K. pneumoniae carbapenemase-2 (KPC-2) in 16 (7.3%) isolates of K. pneumoniae (KPC-2-KP) and IMP-8 in 5 (2.3%) isolates of K. pneumoniae , 5 (33.3%) isolates of E. cloacae , 1 isolate of E. coli , 1 isolate of Klebsiella oxytoca , and one isolate of Citrobacter freundii . The 16 KPC-2-KP isolates were isolated from patients at four different hospitals in northern Taiwan. All 16 of the KPC-2-KP isolates were susceptible to amikacin and colistin and had a similar pulsotype (pulsotype 1) and the same sequence type (sequence type 11). Infections due to KPC-2-KP mainly occurred in severely ill patients in the intensive care unit ( n = 14, 88%). Four patients with infections due to KPC-2-KP died within 14 days of hospitalization. The findings are the first to demonstrate intrahospital and interhospital dissemination of KPC-2-KP in northern Taiwan.

An isolate of Klebsiella oxytoca carrying a novel IMP metallo-β-lactamase was discovered in Madrid, Spain. The bla IMP-28 gene is part of a chromosomally located class I integron. The IMP-28 k cat / K m values for ampicillin, ceftazidime, and cefepime and, to a lesser extent, imipenem and meropenem, are clearly lower than those of IMP-1. The His306Gln mutation may induce important modifications of the L3 loop and thus of substrate accessibility and hydrolysis and be the main reason for this behavior.

We investigated sinks as possible sources of a prolonged Klebsiella pneumonia carbapenemase (KPC)-producing Klebsiella oxytoca outbreak. Seven carbapenem-resistant K. oxytoca isolates were identified in sink drains in 4 patient rooms and in the medication room. Investigations for resistance genes and genetic relatedness of patient and environmental isolates revealed that all the isolates harbored the bla KPC-2 and bla TEM-1 genes and were genetically indistinguishable. We describe here a clonal outbreak caused by KPC-2-producing K. oxytoca , and handwashing sinks were a possible reservoir.

The aim of the present study was to investigate the epidemiological link of multidrug-resistant Klebsiella oxytoca isolates causing community-onset infections among patients attending our outpatient department and to investigate the underlying resistance mechanisms. The isolates were tested by agar dilution MICs, phenotypic carbapenemase testing, enterobacterial repetitive intergenic consensus-PCR, and pulsed-field gel electrophoresis (PFGE). PCR assays and nucleotide sequencing were employed for the identification of bla gene types and the mapping of the integron-containing metallo-β-lactamase (MBL) gene. During the study period (January 2005 to April 2007), nine broad-spectrum cephalosporin-resistant K. oxytoca clinical isolates were prospectively collected from separate outpatients with urinary tract infections. In all cases, the patients had been hospitalized or exposed to health care facilities during the preceding year. Molecular typing revealed that all isolates belonged to the same K. oxytoca clonal type, which contained five PFGE subtypes. A novel chromosomal OXY-2 β-lactamase type variant (OXY-2-9) was detected in all isolates, but no mutations in the promoter region justifying bla OXY gene overproduction were detected. In addition, all isolates harbored the plasmidic CMY-31 (LAT-4) AmpC cephalosporinase, while three of them harbored VIM-1 MBL in a class 1 integron structure. This is the first study to present the dissemination in the community of multidrug-resistant K. oxytoca isolates causing extrahospital infections.

The study shows for the first time the presence of the Klebsiella oxytoca strain fp10 coproducing plasmid-mediated KPC-2 and IMP-8 carbapenemases. The strain was obtained from the fecal sample of an inpatient and showed high-level resistance to imipenem and ertapenem (MICs > 32 μg/ml). Conjugation experiments demonstrated the transferability of the carbapenem-resistant determinants. The results of plasmid analysis and Southern hybridization revealed that the bla KPC-2 gene was located on transferable plasmid pFP10-1 (∼54 kb), whereas the bla IMP-8 gene was on transferable plasmid pFP10-2 (∼180 kb). Analysis of the genetic environment of these two genes has demonstrated that IS Kpn6 and IS Kpn8 are involved in the spread of the bla KPC-2 gene, while the transposable elements IS 26 , intI1 , and tniC might contribute to the dissemination of the bla IMP-8 gene. The chimera of several transposon-associated elements indicated a novel genetic environment of IMP-type metallo-β-lactamase gene in Enterobacteriaceae from China.

The emergence of bla VIM-1 within four different genetic platforms from distinct Enterobacteriaceae and Pseudomonas aeruginosa isolates in an area with a low prevalence of metallo-β-lactamase producers is reported. Forty-three VIM-1-producing isolates (including 19 Enterobacter cloacae , 2 Escherichia coli , and 2 P. aeruginosa isolates, 18 Klebsiella pneumoniae isolate, and 2 Klebsiella oxytoca isolate) recovered from 2005 to 2007 and corresponding to 15 pulsed-field gel electrophoresis types were studied. The Enterobacteriaceae isolates corresponded to a hospital outbreak, and the P. aeruginosa isolates were sporadically recovered. The genetic context of the integrons carrying bla VIM-1 (arbitrarily designated types A, B, C, and D) was characterized by PCR mapping based on known Tn 402 and mercury transposons and further sequencing. Among Enterobacteriaceae isolates, bla VIM-1 was part of integrons located either in an In2-Tn 402 element linked to Tn 21 (type A; In110- bla VIM-1 - aacA4-aadA1 ) or in a Tn 402 transposon lacking the whole tni module [type B; In113- bla VIM-1 - aacA4-dhfrII (also called dfrB1 )- aadA1-catB2 ] and the transposon was associated with an IncHI2 or IncI1 plasmid, respectively. Among P. aeruginosa isolates, bla VIM-1 was part of a new gene cassette array located in a defective Tn 402 transposon carrying either tniB Δ 3 and tniA (type C; bla VIM-1 - aadA1 ) or tniC and Δ tniQ (type D; bla VIM-1 - aadB ), and both Tn 402 variants were associated with conjugative plasmids of 30 kb. The dissemination of bla VIM-1 was associated with different genetic structures and bacterial hosts, depicting a complex emergence and evolutionary network scenario in our facility, Ramón y Cajal University Hospital, Madrid, Spain. Knowledge of the complex epidemiology of bla VIM-1 is necessary to control this emerging threat.

Of 3,004 gram-negative bacilli collected from intra-abdominal infections in the Asia-Pacific region during 2007, 42.2% and 35.8% of Escherichia coli and Klebsiella spp., respectively, were extended-spectrum β-lactamase (ESBL) positive. Moreover ESBL rates in India for E. coli , Klebsiella pneumoniae , and Klebsiella oxytoca were 79.0%, 69.4%, and 100%, respectively. ESBL-positive E. coli rates were also relatively high in China (55.0%) and Thailand (50.8%). Ertapenem and imipenem were the most active drugs tested, inhibiting over 90% of all species, including ESBL-positive isolates with the exception of Pseudomonas aeruginosa isolates (Klebsiella pneumoniae isolates (K. pneumoniae and ESBL-negative K. oxytoca . A decline in ampicillin-sulbactam activity was noted, with only 34.5% of all Enterobacteriaceae inhibited in this study.

Three Klebsiella oxytoca isolates and one Klebsiella pneumoniae isolate from three children admitted to the Hematology Unit of Hospital Vall d'Hebron (Barcelona, Spain) exhibited a susceptibility pattern suggesting OXY β-lactamase hyperproduction. All the isolates contained a 95-kb plasmid that harbored bla OXY-1 , which was transferred by electrotransformation but could not be self-transferred by conjugation. A qnrS1 gene was also harbored in the bla OXY-1 -carrying plasmid. This is the first report of a plasmid-encoded OXY β-lactamase.

Carbapenem-resistant Klebsiella strains carrying Klebsiella pneumoniae carbapenemases (KPC) are endemic to New York City and are spreading across the United States and internationally. Recent studies have indicated that the KPC structural gene is located on a 10-kb plasmid-borne element designated Tn 4401 . Fourteen Klebsiella pneumoniae strains and one Klebsiella oxytoca strain isolated at a New York City hospital in 2005 carrying either bla KPC-2 or bla KPC-3 were examined for isoforms of Tn 4401 . Ten of the Klebsiella strains contained a 100-bp deletion in Tn 4401 , corresponding to the Tn 4401 a isoform. The presence of this deletion adjacent to the upstream promoter region of bla KPC in Tn 4401 a resulted in a different −35 promoter sequence of TGGAGA than that of CTGATT present in isoform Tn 4401 b. Complete sequencing of one plasmid carrying bla KPC from each of three nonclonal isolates indicated the presence of genes encoding other types of antibiotic resistance determinants. The 70.6-kb plasmid from K. pneumoniae strain S9 carrying bla KPC-2 revealed two identical copies of Tn 4401 b inserted in an inverse fashion, but in this case, one of the elements disrupted a group II self-splicing intron. In K. pneumoniae strain S15, the Tn 4401 a element carrying bla KPC-2 was found on both a large 120-kb plasmid and a smaller 24-kb plasmid. Pulsed-field gel electrophoresis results indicate that the isolates studied represent a heterogeneous group composed of unrelated as well as closely related Klebsiella strains. Our results suggest that endemic KPC-positive Klebsiella strains constitute a generally nonclonal population comprised of various alleles of bla KPC on several distinct plasmid genetic backgrounds. This study increases our understanding of the genetic composition of the evolving and expanding role of KPC-producing, healthcare-associated, gram-negative pathogens.

Two CMY-2 derivatives, CMY-31 (Gln 215 →Arg) from Salmonella enterica serotype Newport and CMY-36 (Ala 77 →Cys and Gln 193 →Glu) from Klebsiella pneumoniae , were characterized. Both cephalosporinases functionally resembled CMY-2. bla CMY alleles occurred as parts of a putative transposon comprising IS Ecp1B and a Citrobacter freundii -derived sequence carried by ColE1-like plasmids similar to CMY-5-encoding pTKH11 from Klebsiella oxytoca .

We determined the complete nucleotide sequence of a self-transmissible IncL/M plasmid, pKOI-34, from a Klebsiella oxytoca isolate. pKOI-34 possessed the core structure of an IncL/M plasmid found in Erwinia amylovora , pEL60, with two mobile elements inserted, a transposon carrying the arsenic resistance operon and a Tn 21 -like core module ( tnp and mer modules) piggybacking bla IMP-34 as a class 1 integron, In 808 , where bla IMP-34 confers a resistance to carbapenems in K. oxytoca and Klebsiella pneumoniae .

The activities of tigecycline and comparators against isolates collected from 76 U.S. centers between January 2004 and September 2005 were assessed. Tigecycline MIC 90 s were ≤2 μg/ml for Klebsiella pneumoniae , Klebsiella oxytoca , Escherichia coli , Enterobacter aerogenes , Enterobacter cloacae , Serratia marcescens , Acinetobacter baumannii , Staphylococcus aureus , Enterococcus faecalis , Enterococcus faecium , and Streptococcus agalactiae .

We assessed infections caused by extended-spectrum-β-lactamase-producing Escherichia coli or Klebsiella spp. treated with piperacillin-tazobactam to determine if the susceptibility breakpoint predicts outcome. Treatment was successful in 10 of 11 nonurinary infections from susceptible strains and in 2 of 6 infections with MICs of >16/4 μg/ml. All six urinary infections responded to treatment regardless of susceptibility.

The diversity and evolution of the class A OXY β-lactamase from Klebsiella oxytoca were investigated and compared to housekeeping gene diversity. The entire bla OXY coding region was sequenced in 18 clinical isolates representative of the four K. oxytoca β-lactamase gene groups bla OXY-1 to bla OXY-4 and of two new groups identified here, bla OXY-5 (with four isolates with pI 7.2 and one with pI 7.7) and bla OXY-6 (with four isolates with pI 7.75 and three with pI 8.1). Genes bla OXY-5 and bla OXY-6 showed 99.8% within-group nucleotide similarity but differed from each other by 4.2% and from bla OXY-1 , their closest relative, by 2.5% and 2.9%, respectively. Antimicrobial susceptibility to β-lactams was similar among OXY groups. Nucleotide sequence diversity of the 16S rRNA (1,454 bp), rpoB (940 bp), gyrA (383 bp), and gapDH (573 bp) genes was in agreement with the β-lactamase gene phylogeny. Strains with bla OXY-1 , bla OXY-2 , bla OXY-3 , bla OXY-4 , and bla OXY-6 genes formed five phylogenetic groups, named KoI, KoII, KoIII, KoIV, and KoVI, respectively. Isolates harboring bla OXY-5 appeared to represent an emerging lineage within KoI. We estimated that the bla OXY gene has been evolving within K. oxytoca for approximately 100 million years, using as calibration the 140-million-year estimation of the Escherichia coli - Salmonella enterica split. These results show that the bla OXY gene has diversified along K. oxytoca phylogenetic lines over long periods of time without concomitant evolution of the antimicrobial resistance phenotype.

We investigated a Klebsiella oxytoca isolate demonstrating resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The MICs of both imipenem and meropenem were 32μ g/ml. The β-lactamase activity against imipenem and meropenem was inhibited in the presence of clavulanic acid. Isoelectric focusing studies demonstrated five β-lactamases with pIs of 8.2 (SHV-46), 6.7 (KPC-2), 6.5 (unknown), 6.4 (probable OXY-2), and 5.4 (TEM-1). The presence of the bla SHV and bla TEM genes was confirmed by specific PCR assays and DNA sequence analysis. Transformation and conjugation studies with Escherichia coli showed that the β-lactamase with a pI of 6.7, Klebsiella pneumoniae carbapenemase-2 (KPC-2), was encoded on an approximately 70-kb conjugative plasmid that also carried SHV-46, TEM-1, and the β-lactamase with a pI of 6.5. The bla KPC-2 determinant was cloned in E. coli and conferred resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The amino acid sequence of KPC-2 showed a single amino acid difference, S174G, when compared with KPC-1, another carbapenem-hydrolyzing β-lactamase from K. pneumoniae 1534. Hydrolysis studies showed that purified KPC-2 hydrolyzed not only carbapenems but also penicillins, cephalosporins, and aztreonam. KPC-2 had the highest affinity for meropenem. The kinetic studies revealed that KPC-2 was inhibited by clavulanic acid and tazobactam. An examination of the outer membrane proteins of the parent K. oxytoca strain demonstrated that it expressed detectable levels of OmpK36 (the homolog of OmpC) and a higher-molecular-weight OmpK35 (the homolog of OmpF). Thus, carbapenem resistance in K. oxytoca 3127 is due to production of the Bush group 2f, class A, carbapenem-hydrolyzing β-lactamase KPC-2. This β-lactamase is likely located on a transposon that is part of a conjugative plasmid and thus has a very high potential for dissemination.

The two genetic groups (oxy-1 and oxy-2) previously identified in the Klebsiella oxytoca taxon are recognizable by four independent molecular markers: (i) ERIC-1R profiles, (ii) 16S ribosomal DNA (rDNA) signature sequences, (iii) singular nucleotides in a defined fragment of the rpoB gene, and (iv) the type of the strain's bla OXY gene (i.e., bla OXY-1 or bla OXY-2 ). K. oxytoca strains SG266 and SG271 could not be classified into these genetic groups based on their ERIC-1R profile and bla OXY gene sequence. With regard to the gene identity percentages between the bla OXY-1 and bla OXY-2 gene groups (86.8% ± 0.4%) and within a bla OXY gene group (>99%), it was concluded that the bla OXY gene of strain SG271 was representative of a new bla OXY gene group ( bla OXY-3 ), since the mean identity percentages between it and the two bla OXY gene groups were 85.5% ± 0.2% and 84.4% ± 0.4%, respectively. Since the corresponding percentages were 95.0% ± 0.4% and 86.2% ± 0.3% for strain SG266, it was impossible to classify its bla OXY gene, which was therefore named bla OXY-4 . The 16S rDNA signature sequences of the two strains could be determined only after cloning experiments. The SG266 clones displayed the same signature sequence as that of the genetic group oxy-1, whereas the SG271 clones displayed three different 16S rDNA signature sequences that also differed from those of the two genetic groups. Singular nucleotides were found within the rpoB sequence of the two strains, allowing for their distinction from the two genetic groups. All of these results, combined with those previously obtained by the ERIC-1R PCR method, indicate that strain SG271 is representative of a new K. oxytoca genetic group (oxy-3), whereas strain SG266 could not be classified.

From whole-cell DNA of an enterobacterial Erwinia persicina reference strain that displayed a penicillinase-related antibiotic-resistant phenotype, a β-lactamase gene was cloned and expressed in Escherichia coli. It encoded a clavulanic-acid-inhibited Ambler class A β-lactamase, ERP-1, with a pI value of 8.1 and a relative molecular mass of ca. 28 kDa. ERP-1 shared 45 to 50% amino acid identity with the most closely related enzymes, the chromosomally encoded enzymes from Citrobacter koseri , Kluyvera ascorbata , Kluyvera cryocrescens , Klebsiella oxytoca , Proteus vulgaris , Proteus penneri , Rahnella aquatilis , Serratia fonticola , Yersinia enterocolitica , and the plasmid-mediated enzymes CTX-M-8 and CTX-M-9. The substrate profile of the noninducible ERP-1 was similar to that of these β-lactamases. ERP-1 is the first extended-spectrum β-lactamase from an enterobacterial species that is plant associated and plant pathogenic.

The emergence and spread of New Delhi metallo-β-lactamase 1 (NDM-1)-producing carbapenem-resistant Enterobacteriaceae (CRE) present an urgent threat to human health. In China, the bla NDM-1 gene has been reported mostly in Acinetobacter spp. but is rarely found in Enterobacteriaceae . Here, we report a high incidence and endemic spread of NDM-1-producing CRE in Henan Province in China. Sixteen (33.3%) of the 48 CRE isolates obtained from patients during June 2011 to July 2012 were positive for bla NDM-1 , and the gene was found to be carried on plasmids of various sizes (∼55 to ∼360 kb). These plasmids were readily transferrable to recipient Escherichia coli by conjugation, conferred resistance to multiple antibiotics, and belonged to multiple replicon types. The bla NDM-1 -positive CRE isolates were genetically diverse, and six new multilocus sequence typing (MLST) sequence types were linked to the carriage of NDM-1. Five of the isolates were classified as extensively drug-resistant (XDR) isolates, four of which also carried the fosA3 gene conferring resistance to fosfomycin, an alternative drug for treating infections by CRE. In each bla NDM-1 -positive CRE isolate, the bla NDM-1 gene was downstream of an intact IS Aba125 element and upstream of the ble MBL gene. Furthermore, gene environment analysis suggested the possible transmission of bla NDM-1 -containing sequences from Acinetobacter spp. to Klebsiella pneumoniae and Klebsiella oxytoca . These findings reveal the emergence and active transmission of NDM-1-positive CRE in China and underscore the need for heightened measures to control their further spread.

Citrobacter sedlakii 2596, a clinical strain resistant to aminopenicillins, carboxypenicillins, and early cephalosporins such as cephalothin, but remaining susceptible to acylureidopenicillins, carbapenems, and later cephalosporins such as cefotaxime, was isolated from the bile of a patient treated with β-lactam and quinolone antibiotics. The isolate produced an inducible class A β-lactamase of pI 8.6, named Sed-1, which was purified. Characterized by a molecular mass of 30 kDa, Sed-1 preferentially hydrolyzed benzylpenicillin, cephalothin, and cloxacillin. The corresponding gene, bla Sed-1 , was cloned and sequenced. Its deduced amino acid sequence shared more than 60% identity with the chromosome-encoded β-lactamases from Citrobacter koseri (formerly C. diversus ) (84%), Klebsiella oxytoca (74%), Serratia fonticola (67%), and Proteus vulgaris (63%) and 71% identity with the plasmid-mediated enzyme MEN-1. A gene coding for a LysR transcriptional regulator was found upstream from bla Sed-1 . This regulator, named SedR, displayed 90% identity with the AmpR sequence of the chromosomal β-lactamase from C. koseri and 63 and 50% identity with the AmpR sequences of P. vulgaris and Enterobacter cloacae , respectively. By using DNA-DNA hybridization, a bla Sed-1 -like gene was identified in two reference strains, C. sedlakii (CIP-105037) and Citrobacter rodentium (CIP-104675), but not in the 18 strains of C. koseri studied. Two DNA fragments were amplified and sequenced from the reference strains of C. sedlakii CIP-105037 and C. rodentium CIP-104675 using two primers specific for bla Sed-1 . They shared 98 and 80% identity with bla Sed-1 , respectively, confirming the diversity of the chromosomally encoded class A β-lactamases found in Citrobacter .

A transferable β-lactamase produced by a multidrug-resistant clinical isolate of Enterobacter cloacae was studied. The bla gene was carried by a large (>80-kb) transmissible plasmid. Nucleotide sequence analysis of cloned fragments revealed that it was part of a gene cassette carried by a class 1 integron along with other resistance genes, including aac ( 6 ′) -Ib . The encoded β-lactamase, designated IBC-1, was a novel class A enzyme that hydrolyzed ceftazidime and cefotaxime and was inhibited by tazobactam and, to a lesser extent, by clavulanate. Also, imipenem exhibited potent inhibitory activity against IBC-1. The enzyme consisted of 287 amino acid residues, including Ser-237, cysteines at positions 69 and 237a, and Arg-244, which may be implicated in its interaction with β-lactams. In amino acid sequence comparisons, IBC-1 displayed the highest similarity with the chromosomal penicillinase of Yersinia enterocolitica , a carbenicillinase from Proteus mirabilis GN79, the species-specific β-lactamases of Klebsiella oxytoca , and the carbapenemase Sme-1. However, a phylogenetic association with established β-lactamase clusters could not be conclusively shown.

The in vitro activities of ciprofloxacin, clinafloxacin, gatifloxacin, levofloxacin, moxifloxacin, and trovafloxacin were tested against 72 ciprofloxacin-resistant and 28 ciprofloxacin-susceptible isolates of Klebsiella pneumoniae , Klebsiella oxytoca , Enterobacter cloacae , and Enterobacter aerogenes . Irrespective of the alterations in GyrA and ParC proteins, clinafloxacin exhibited greater activity than all other fluoroquinolones tested against K. pneumoniae and E. aerogenes .

A clinical isolate of Klebsiella oxytoca (Kox 443) was found to have a low-level resistance to broad-spectrum penicillins (MICs of amoxicillin and ticarcillin, 256 and 32 μg/ml, respectively), without substantial potentiation by 2 μg of clavulanic acid per ml (amoxicillin- and ticarcillin-clavulanate, 128 and 8 μg/ml, respectively), while being fully susceptible to cephalosporins and other β-lactam antibiotics. These resistances were carried by a ca. 50-kb conjugative plasmid that encodes a single β-lactamase with a pI of 5.6. Compared to TEM-2, this enzyme exhibited a 3- to 30-fold higher K m and a decreased maximal hydrolysis rate for β-lactams; higher concentrations of suicide inactivators (5- to 500-fold higher concentrations giving a 50% reduction in hydrolysis) were required for inhibition. Nucleotide sequence analysis revealed identity between the bla TEM gene of Kox 443 and the bla TEM-2 gene, except for a single A-to-G change at position 590, leading to the amino acid change from Ser-130 Gly. This mutation has not been reported previously in the TEM type β-lactamases produced by clinical strains, and the novel enzyme was called TEM-59 (alternative name IRT-17). This is the first description of an inhibitor-resistant TEM-derived enzyme in the species K. oxytoca.

Plasmid pTKH11, originally obtained by electroporation of a Klebsiella oxytoca plasmid preparation into Escherichia coli XAC, expressed a high level of an AmpC-like β-lactamase. The enzyme, designated CMY-5, conferred resistance to extended-spectrum β-lactams in E. coli ; nevertheless, the phenotype was cryptic in the K. oxytoca donor. Determination of the complete nucleotide sequence of pTKH11 revealed that the 8,193-bp plasmid encoded seven open reading frames, including that for the CMY-5 β-lactamase ( bla CMY-5 ). The bla CMY-5 product was similar to the plasmidic CMY-2 β-lactamase of K. pneumoniae and the chromosomal AmpC of Citrobacter freundii , with 99.7 and 97.0% identities, respectively; there was a substitution of phenylalanine in CMY-5 for isoleucine 105 in CMY-2. bla CMY-5 was followed by the Blc and SugE genes of C. freundii , and this cluster exhibited a genetic organization identical to that of the ampC region on the chromosome of C. freundii ; these results confirmed that C. freundii AmpC was the evolutionary origin of the plasmidic cephamycinases. In the K. oxytoca host, the copy number of pTKH11 was very low and the plasmid coexisted with plasmid pNBL63. Analysis of the replication regions of the two plasmids revealed 97% sequence similarity in the RNA I transcripts; this result implied that the two plasmids might be incompatible. Incompatibility of the two plasmids might explain the cryptic phenotype of bla CMY-5 in K. oxytoca through an exclusion effect on pTKH11 by resident plasmid pNBL63.

Two β-lactamase gene regions were characterized by DNA sequencing in eight clinical isolates of Klebsiella oxytoca . The bla OXY-2a region encoded a β-lactamase nearly identical to OXY-2 (one amino acid residue substituted) and conferred aztreonam and cefuroxime resistance on the K. oxytoca isolates. Overproduction of OXY-2a was caused by a G-to-A substitution of the fifth nucleotide in the −10 consensus sequence of bla OXY-2a . The bla OXY-1a was identified in a susceptible strain, and the OXY-1a enzyme differed from OXY-1 by two amino acid residues.

Fluoroquinolone resistance (FQ-R) in clinical isolates of Enterobacteriaceae species has been reported with increasing frequency in recent years. Two mechanisms of FQ-R have been identified in gram-negative organisms: mutations in DNA gyrase and reduced intracellular drug accumulation. A single point mutation in gyrA has been shown to reduce susceptibility to fluoroquinolones. To determine the extent of gyrA mutations associated with FQ-R in enteric bacteria, one set of oligonucleotide primers was selected from conserved sequences in the flanking regions of the quinolone resistance-determining regions (QRDR) of Escherichia coli and Klebsiella pneumoniae . This set of primers was used to amplify and sequence the QRDRs from 8 Enterobacteriaceae type strains and 60 fluoroquinolone-resistant clinical isolates of Citrobacter freundii , Enterobacter aerogenes , Enterobacter cloacae , E. coli , K. pneumoniae , Klebsiella oxytoca , Providencia stuartii , and Serratia marcescens . Although similarity of the nucleotide sequences of seven species ranged from 80.8 to 93.3%, when compared with that of E. coli , the amino acid sequences of the gyrA QRDR were highly conserved. Conservative amino acid substitutions were detected in the QRDRs of the susceptible type strains of C. freundii , E. aerogenes , K. oxytoca (Ser-83 to Thr), and P. stuartii (Asp-87 to Glu). Strains with ciprofloxacin MICs of >2 μg/ml expressed amino acid substitutions primarily at the Gly-81, Ser-83, or Asp-87 position. Fluoroquinolone MICs varied significantly for strains exhibiting identical gyrA mutations, indicating that alterations outside gyrA contribute to resistance. The type and position of amino acid alterations also differed among these six genera. High-level FQ-R frequently was associated with single gyrA mutations in all species of Enterobacteriaceae in this study except E. coli.

The sequence of the gene encoding a novel cefotaxime-hydrolyzing β-lactamase (CTX-M-4) was determined. It was located in a plasmid harbored by a Salmonella typhimurium strain. CTX-M-4 was similar to the plasmidic cefotaxime-hydrolyzing β-lactamases CTX-M-2 and Toho-1 and related to the chromosomal β-lactamase of Klebsiella oxytoca . A Ser-237→Ala substitution, introduced by site-directed mutagenesis, caused minor alterations in the interaction of CTX-M-4 with β-lactams, reducing slightly the relative hydrolytic activity against cefotaxime and the susceptibility to inhibition by clavulanate.

Fecal Klebsiella isolates from neonates in 22 Swedish special care units were examined by a PCR we developed for detection of the SHV-1 beta-lactamase gene. All 105 K. pneumoniae isolates and all 11 K. pneumoniae reference strains (including the K. pneumoniae subsp. pneumoniae, ozaenae, and rhinoscleromatis type strains) tested were positive, whereas all 67 K. oxytoca isolates and the K. oxytoca, K. planticola, and K. terrigena type strains tested were negative. Resistance to beta-lactams in K. pneumoniae was not transferable by conjugation, and the beta-lactamase gene was never found on a plasmid. Southern blot analysis showed that the gene had a defined chromosomal location. Isoelectric focusing and sequencing of 231-bp PCR amplicons from different isolates revealed many variants of the enzyme, with the two main groups being SHV-1 like (pI 7.6; 68 isolates) and LEN-1 like (pI 7.1; 14 isolates). Clavulanic acid markedly reduced the MICs of ampicillin for all the K. pneumoniae isolates tested. This fact, MIC profiles (penicillin rather than cephalosporin resistance), pIs, and sequence data showed that the chromosomal beta-lactamase of K. pneumoniae is a class A, group 2 enzyme distinct from the chromosomal AmpC enzymes found in several other gram-negative bacteria and from the chromosomal beta-lactamase K1 of K. oxytoca. We propose that the chromosomal beta-lactamase of K. pneumoniae be designated K2 and suggest that an allelic pI 7.6 variant of this enzyme is the ancestor of the SHV family of plasmid-mediated beta-lactamases.

The beta-lactamase genes of Klebsiella oxytoca were previously divided into two main groups: bla(OXY-1) and bla(OXY-2). The two beta-lactamase groups were each represented by beta-lactamases with four different pIs. In each group, one form of beta-lactamase is more frequent than the others combined. The beta-lactamase gene of each representative beta-lactamase with a different pI that was not yet sequenced (pIs 5.7, 6.8 [OXY-2], 7.1, 8.2, and 8.8 [OXY-1]) was cloned and sequenced. The susceptibility patterns as well as relative rates and kinetic parameters for beta-lactam hydrolysis revealed that OXY-2 enzymes hydrolyzed several of the beta-lactams that were examined (carbenicillin, cephalothin, cefamandole, ceftriaxone, and aztreonam) at a greater rate than the OXY-1 enzymes did. Comparison of K. oxytoca beta-lactamases with plasmid-mediated extended-spectrum beta-lactamases MEN-1 and TOHO-1 implied that the threonine at position 168 present in OXY-2 beta-lactamase instead of the alanine in OXY-1 could be responsible for its modified substrate hydrolysis. In each group, the beta-lactamase with a variant pI differs from the main form of beta-lactamase by one to five amino acid substitutions. The substrate profile and the 50% inhibitory concentrations revealed that all substitutions differing from the main form of beta-lactamase were neutral except one difference in the OXY-1 group. This substitution of an Ala to a Gly at position 237 increases the hydrolysis of some beta-lactams, particularly aztreonam; decreases the hydrolysis of benzylpenicillin, cephaloridine, and cefamandole, and decreases the susceptibility to clavulanic acid (fivefold increase in the 50% inhibitory concentration).

Escherichia coli (328 isolates), Klebsiella pneumoniae (296), Klebsiella oxytoca (44), and Proteus mirabilis (33) isolates collected during 2012 from the nine U.S. census regions and displaying extended-spectrum-β-lactamase (ESBL) phenotypes were evaluated for the presence of β-lactamase genes, and antimicrobial susceptibility profiles were analyzed. The highest ESBL rates were noted for K. pneumoniae (16.0%, versus 4.8 to 11.9% for the other species) and in the Mid-Atlantic and West South Central census regions. CTX-M group 1 (including CTX-M-15) was detected in 303 strains and was widespread throughout the United States but was more prevalent in the West South Central, Mid-Atlantic, and East North Central regions. KPC producers (118 strains [112 K. pneumoniae strains]) were detected in all regions and were most frequent in the Mid-Atlantic region (58 strains). Thirteen KPC producers also carried bla CTX-M . SHV genes encoding ESBL activity were detected among 176 isolates. Other β-lactamase genes observed were CTX-M group 9 (72 isolates), FOX (10), TEM ESBL (9), DHA (7), CTX-M group 2 (3), NDM-1 (2 [Colorado]), and CTX-M groups 8 and 25 (1). Additionally, 62.9% of isolates carried ≥2 β-lactamase genes. KPC producers were highly resistant to multiple agents, but ceftazidime-avibactam (MIC 50/90 , 0.5/2 μg/ml) and tigecycline (MIC 50/90 , 0.5/1 μg/ml) were the most active agents tested. Overall, meropenem (MIC 50 , ≤0.06 μg/ml), ceftazidime-avibactam (MIC 50 , 0.12 to 0.5 μg/ml), and tigecycline (MIC 50 , 0.12 to 2 μg/ml) were the most active antimicrobials when tested against this collection. NDM-1 producers were resistant to all β-lactams tested. The diversity and increasing prevalence of β-lactamase-producing Enterobacteriaceae have been documented, and ceftazidime-avibactam was very active against the vast majority of β-lactamase-producing strains isolated from U.S. hospitals.

Since the first isolation in 2002, the metallo-β-lactamase GIM-1 has not been detected outside Germany. The data presented here, for 50 clinical bla GIM-1 -positive isolates, including Pseudomonas spp. and Enterobacteriaceae ( Enterobacter cloacae , Klebsiella oxytoca , Serratia marcescens , Escherichia coli , and Citrobacter freundii ), collected between 2007 and 2012 at the original site in an ongoing outbreak, demonstrate a diverse genetic background and dissemination of the gene conferring resistance to enteric bacteria.

Nine Klebsiella oxytoca strains which demonstrated resistance to the combination of sulbactam and cefoperazone were isolated from geographically separate hospitals in Japan in 1995. Among them, K. oxytoca SB23 showed high-level resistance to sulbactam-cefoperazone (MIC > 128 micrograms/ml) and aztreonam (MIC, 128 micrograms/ml). The sulbactam-cefoperazone resistance was not transferred from strain SB23 to Escherichia coli CSH2 by conjugation, beta-Lactamase RbiA, produced by strain SB23, was purified, and the molecular mass was estimated to be 29 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinetic parameters for RbiA revealed that cefoperazone and aztreonam were hydrolyzed efficiently by this enzyme. Moreover, ceftazidime and imipenem were also hydrolyzed weakly by RbiA, although strain SB23 did not show any resistance to these agents. Clavulanate, sulbactam, and tazobactam failed to block the hydrolysis of cefoperazone by RbiA. The structural gene of RbiA (blaRBI) was cloned and sequenced, and the deduced amino acid sequence of RbiA demonstrated high-level similarities to those of the beta-lactamases found in K. oxytoca D488, E23004, and plasmid-mediated MEN-1, which have been classified into Bush functional group 2be. Although RbiA demonstrates high-level molecular similarity to the enzymes in group 2be, from an enzymological point of view, this enzyme might be differentiated from the enzymes in that group. Hybridization analysis revealed that beta-lactamase genes highly similar to blaRBI were generally encoded on the chromosome of the sulbactam-cefoperazone-resistant clinical isolates of K. oxytoca tested in the study, despite their different derivations. This observation suggests that sulbactam-cefoperazone-resistant A. oxytoca strains which produce RbiA-type beta-lactamases have been proliferating in many hospitals in Japan.

Amino acid sequences determined either by protein sequencing or by DNA sequencing are identical for cefotaximases CTX-M-1 and MEN-1, whereas CTX-M-2 is 84% identical to CTX-M-1/MEN-1. Both beta-lactamases are distantly related to other plasmidic class A enzymes (homology to TEM-1 is 38.1% for CTX-M-1/MEN-1 and 36.5% for CTX-M-2); the closest relationship was with the chromosomal beta-lactamase of Klebsiella oxytoca E23004 (homologies of 74.5% for CTX-M-1/MEN-1 and 77.9% for CTX-M-2). The cefotaximases CTX-M-1/MEN-1 and CTX-M-2 represent two members of a new subgroup of plasmidic class A beta-lactamases.

The chromosomally encoded beta-lactamase gene (blaOXY-2) of the wild-type Klebsiella oxytoca SL911 was cloned and sequenced. Its nucleotide sequence similarity with the previously sequenced K. oxytoca beta-lactamase gene (blaOXY-1) (Y. Arakawa, M. Ohta, N. Kido, M. Mori, H. Ito, T. Komatsu, Y. Fujii, and N. Kato, Antimicrob. Agents Chemother. 33:63-70, 1989) is 87.3%, and its amino acid similarity is 89.7%. This group of K. oxytoca beta-lactamases is related to chromosomal beta-lactamases of Citrobacter diversus, Proteus vulgaris, and Yersinia enterocolitica and to the plasmid-mediated extended-spectrum beta-lactamases MEN-1 and Toho-1. By colony hybridization with 86 strains susceptible and resistant to aztreonam, isolated in six countries, K. oxytoca beta-lactamase genes hybridized with either a specific blaOXY-1 DNA probe (668 bp) or a blaOXY-2 DNA probe (723 bp). Thus, beta-lactamase genes could be divided into two groups: blaOXY-1 (47% of the strains) and blaOXY-2 (53% of the strains). A study of isoelectric points confirmed the great variability reported in the literature. However, the two beta-lactamase groups were each represented by four different pIs: for OXY-2, 5.2, 5.7, 6.4, and 6.8, with the 5.2 form representing 59% of all OXY-2 enzymes, and for OXY-1, 7.1, 7.5, 8.2, and 8.8, with the 7.5 form representing 88% of all OXY-1 enzymes.