Jiancheng Zhang

Activities of NXL104 Combinations with Ceftazidime and Aztreonam against Carbapenemase-Producing Enterobacteriaceae

ABSTRACT Combinations of NXL104 with ceftazidime and aztreonam were tested against carbapenem-resistant members of the Enterobacteriaceae. Ceftazidime-NXL104 was active against strains with the OXA-48 enzyme or with combinations of impermeability and an extended-spectrum β-lactamase (ESBL) or AmpC enzyme and also against most Klebsiella spp. with the KPC enzyme, but metallo-β-lactamase producers were resistant. Aztreonam-NXL104 was active against all carbapenemase producers at 4 and 4 μg/ml, including those with metallo-β-lactamases.

Activity of aminoglycosides, including ACHN-490, against carbapenem-resistant Enterobacteriaceae isolates

BACKGROUND the emergence of carbapenemases in Enterobacteriaceae is driving a search for therapeutic alternatives. We tested ACHN-490, a sisomicin derivative that evades all plasmid-mediated aminoglycoside-modifying enzymes, against 82 carbapenem-resistant Enterobacteriaceae isolates. Comparators included internationally and locally available aminoglycosides. Methods The isolates variously had KPC (n = 12), SME-1 (n = 1), IMP (n = 13), VIM (n = 5), NDM (n = 17) or OXA-48 (n = 19) carbapenemases, or had combinations of impermeability with AmpC (n = 5) or extended-spectrum β-lactamases (n = 10). They included 53 Klebsiella spp., 19 Enterobacter spp., 6 Escherichia coli and 4 others; most were multiresistant. Genes were identified by PCR and sequencing; MICs were measured by CLSI agar dilution. RESULTS ACHN-490 was active at ≤ 2 mg/L against all 65 isolates with carbapenem resistance mechanisms other than NDM enzyme, mostly with MICs of 0.12-0.5 mg/L; isepamicin was active against 63/65 at ≤ 8 mg/L. In contrast, 35% were resistant to gentamicin at 4 mg/L, 61% to tobramycin at 4 mg/L and 20% to amikacin at 16 mg/L. However, 16 of the 17 isolates with NDM-1 enzyme were resistant to ACHN-490, with MICs ≥ 64 mg/L, and these were cross-resistant to all other human-use aminoglycosides tested. Their behaviour was associated with ArmA and RmtC 16S rRNA methylases. Apramycin (a veterinary aminoglycoside) retained its full activity, with MICs of 4-8 mg/L versus strains with armA or rmtC, though resistance was seen in one Klebsiella pneumoniae with AAC(3)-IV (MIC ≥ 256 mg/L). CONCLUSIONS ACHN-490 has potent activity versus carbapenem-resistant isolates, except those also harbouring 16S rRNA methylases; isepamicin is also widely active, though less potent than ACHN-490. Evasion of 16S rRNA methylases by apramycin is noteworthy and may provide a starting point for future aminoglycoside development.

Prevalence of faecal carriage of Enterobacteriaceae with NDM-1 carbapenemase at military hospitals in Pakistan, and evaluation of two chromogenic media

OBJECTIVES To determine the prevalence and antimicrobial susceptibility of carbapenemase-producing Enterobacteriaceae among hospitalized patients and outpatients attending two military hospitals in Rawalpindi, Pakistan, and to compare the performance of two chromogenic culture media for the isolation of these organisms. METHODS Stool samples from 200 distinct patients were cultured on MacConkey agar and subsequently on two chromogenic media-Colorex KPC and a prototype chromogenic medium, ID Carba-designed for the isolation of carbapenemase-producing Enterobacteriaceae. All Gram-negative isolates growing on either chromogenic medium were investigated for carbapenemases by phenotypic and molecular methods. Producers were subjected to susceptibility testing with 40 antimicrobials by VITEK 2 or agar dilution. RESULTS In total, 64 NDM-1-positive isolates of Enterobacteriaceae, belonging to seven distinct species, were recovered from 37 (18.5%) of the stool samples. No other carbapenemase types were confirmed. Nineteen positive samples were identified among 70 from inpatients (prevalence 27.1%) and there were 18 positive samples among 130 from outpatients (prevalence 13.8%). Fifty-six isolates (87.5%) harbouring the NDM-1 enzyme were recovered on ID Carba compared with 41 isolates (64.1%) on Colorex KPC (P = 0.012). Multidrug resistance was prevalent, but no pan-resistant isolates were found, with most isolates susceptible in vitro to colistin (97%), mecillinam (95%), fosfomycin (94%), tigecycline (89%) and nitrofurantoin (78%). CONCLUSIONS This study shows a high prevalence of multidrug-resistant Enterobacteriaceae with the NDM-1 enzyme in Rawalpindi. The new chromogenic medium, ID Carba, was more sensitive than Colorex KPC and has potential as a screening medium for isolation of Enterobacteriaceae harbouring the NDM-1 enzyme.

Arrival of Klebsiella pneumoniae producing KPC carbapenemase in the United Kingdom

BACKGROUND KPC-type carbapenemases are increasingly prevalent in parts of the USA and Israel and are an emerging concern in South America, Europe and China. We investigated the UKs first two KPC-producing Klebsiella pneumoniae isolates. METHODS The isolates were referred to the UKs national reference laboratory for confirmation of carbapenem resistance. Susceptibilities were determined by agar dilution, and bla(KPC) and Tn4401-like elements were sought by PCR and sequencing. Isolates were compared by PFGE of XbaI- and SpeI-digested genomic DNA. RESULTS The isolates were from patients in different UK hospitals, with no epidemiological connection. Both were resistant to carbapenems (MICs > 16 mg/L), with imipenem MICs unchanged by EDTA, and also to all other beta-lactams (including inhibitor combinations), tobramycin, amikacin and ciprofloxacin. They were susceptible to gentamicin (MICs </= 1 mg/L) and colistin (MICs </= 0.5 mg/L), with intermediate susceptibility to tigecycline (MICs 1-2 mg/L). The isolates belonged to the same PFGE-defined strain, highly related to a disseminated KPC-producing strain characterized previously in Tel Aviv, Israel. Like this Israeli strain, the UK isolates produced KPC-3 carbapenemase, with the bla(KPC-3) gene located within a Tn4401-like element. CONCLUSIONS The first KPC-3-producing K. pneumoniae isolates detected in the UK were highly genetically related to a KPC-3-producing Israeli K. pneumoniae strain. This relatedness was consistent with the history of one UK patient, who had been hospitalized previously in Israel. However, this strain may be circulating more widely since the second UK patient had no identifiable links with Israel or other overseas countries.

Comparison of BD Phoenix, Vitek 2, and MicroScan Automated Systems for Detection and Inference of Mechanisms Responsible for Carbapenem Resistance in Enterobacteriaceae

ABSTRACT We assessed the ability of three commercial systems to infer carbapenem resistance mechanisms in 39 carbapenemase-producing and 16 other carbapenem-resistant Enterobacteriaceae. The sensitivity/specificity values for “flagging” a likely carbapenemase were 100%/0% (BD Phoenix), 82 to 85%/6 to 19% (MicroScan), and 74%/38% (Vitek 2), respectively. OXA-48 producers were poorly detected, but all systems reliably detected isolates with KPC and most with metallo-carbapenemases.

Evaluation of an expanded microarray for detecting antibiotic resistance genes in a broad range of Gram-negative bacterial pathogens.

ABSTRACT A microarray capable of detecting genes for resistance to 75 clinically relevant antibiotics encompassing 19 different antimicrobial classes was tested on 132 Gram-negative bacteria. Microarray-positive results correlated >91% with antimicrobial resistance phenotypes, assessed using British Society for Antimicrobial Chemotherapy clinical breakpoints; the overall test specificity was >83%. Microarray-positive results without a corresponding resistance phenotype matched 94% with PCR results, indicating accurate detection of genes present in the respective bacteria by microarray when expression was low or absent and, hence, undetectable by susceptibility testing. The low sensitivity and negative predictive values of the microarray results for identifying resistance to some antimicrobial resistance classes are likely due to the limited number of resistance genes present on the current microarray for those antimicrobial agents or to mutation-based resistance mechanisms. With regular updates, this microarray can be used for clinical diagnostics to help accurate therapeutic options to be taken following infection with multiple-antibiotic-resistant Gram-negative bacteria and prevent treatment failure.

Detection of Pseudomonas aeruginosa isolates producing VEB-type extended-spectrum β-lactamases in the United Kingdom

OBJECTIVES The aim of this study was to investigate the presence of VEB enzymes among Pseudomonas spp. referred to the UKs national reference laboratory and with phenotypic evidence of extended-spectrum beta-lactamase (ESBL) production. METHODS Antibiograms were analysed for Pseudomonas spp. referred from November 2003 to November 2007. Isolates with >/=4-fold ceftazidime/clavulanate synergy were screened for bla(VEB) alleles. Genes encoding metallo-beta-lactamases (bla(MBL)) were sought in isolates with positive imipenem/EDTA synergy tests. Selected PCR products were sequenced. PFGE of SpeI-digested genomic DNA was used to compare isolates. RESULTS Forty-nine (3.7%) of 1338 Pseudomonas spp. were considered potential ESBL producers; 40 were recovered for molecular testing. bla(VEB) alleles were detected in 32 Pseudomonas aeruginosa isolates, comprising diverse PFGE types, from 12 UK hospitals and 1 in India. One UK centre referred 15 isolates with VEB-1 enzyme; these were serotype O15, representing a single PFGE-defined strain that also produced VIM-10 metallo-carbapenemase. This strain was resistant to all beta-lactams, aminoglycosides and ciprofloxacin, remaining susceptible only to colistin (MICs </=1 mg/L). Two other P. aeruginosa isolates co-producing both VEB and VIM enzymes were received from two other UK hospitals; one isolate represented inter-hospital spread of the O15 strain and the second was distinct. CONCLUSIONS VEB enzymes have not been reported previously in the UK, but were produced by 80% of Pseudomonas spp. with phenotypic evidence of ESBL production. They co-existed with VIM carbapenemases in two strains, with one responsible for a major hospital outbreak. The incidence of ESBLs may be underestimated in Pseudomonas because ESBL phenotypes can be masked by other beta-lactam resistance mechanisms.

Evaluation of a commercial microarray to detect carbapenemase-producing Enterobacteriaceae

Sir, Enterobacteriaceae with extended-spectrum b-lactamases (ESBLs) are a global problem and their rising prevalence has resulted in increased use of carbapenem antibiotics. Consequently, many countries are now detecting growing numbers of carbapenem-resistant Enterobacteriaceae, with resistance mediated by ESBLs and/or AmpC enzymes in combination with reduced permeability due to mutations that cause porin loss, or by true carbapenemases. These carbapenemases are diverse and variously belong to b-lactamase molecular groups A (e.g. KPC types), B (e.g. IMP, VIM and NDM types) and D (e.g. OXA-48). Prompt detection of carbapenemase-producing bacteria is essential for implementation of the infection control procedures necessary to limit their spread, and may also guide individual patient management (http://www.hpa.org.uk). Various molecular methodologies have been devised for rapid gene detection, and microarrays offer the advantage of detecting multiple genes in a single test. We evaluated the commercial Check-MDR CT102 ESBL-Carbapenemase microarray (Check-Points Health BV, Wageningen, The Netherlands) to detect genes encoding carbapenemases in 57 clinical isolates of Enterobacteriaceae (Table 1). Many of these isolates had been referred to the HPA’s Antibiotic Resistance Monitoring and Reference Laboratory for investigation of carbapenem resistance, and they included 41 known to have a carbapenemase and 16 that were carbapenem-resistant through a combination of ESBL/AmpC activity plus impermeability (porin loss); 7 carbapenem-susceptible controls were also tested. Most isolates were tested against this commercial array only once, since this is how they would be used in practice, unless there was a discrepancy between the expected and array results. Total bacterial DNA was extracted from the isolates using a Blood and Tissue DNA Purification Kit (QIAGEN, Crawley, UK). Undiluted DNA (10 mL) was used as template in assays undertaken according to the manufacturer’s instructions. The principle and molecular components of these array-based assays have

Dissemination of pCT-Like IncK Plasmids Harboring CTX-M-14 Extended-Spectrum β-Lactamase among Clinical Escherichia coli Isolates in the United Kingdom

ABSTRACT IncK plasmids encoding CTX-M-14 extended-spectrum β-lactamase (ESBL) and highly related to plasmid pCT were detected in 13 of 67 (19%) human clinical isolates of Escherichia coli with a group 9 CTX-M-type ESBL from the United Kingdom and in 2 quality assurance isolates. None of these E. coli strains was related to the cattle strain from which pCT was originally characterized.

IMP metallo-β-lactamase-producing clinical isolates of Enterobacter cloacae in the UK

Sir, Carbapenems, such as imipenem, meropenem and ertapenem, are often the main option for treatment of infections caused by multiresistant Gram-negative bacteria, particularly those that produce extended-spectrum or AmpC b-lactamase enzymes. However, the clinical utility of these antimicrobials is under threat with the emergence and spread of acquired genes coding for carbapenem-hydrolysing b-lactamases. These enzymes are broadly classified as serine carbapenemases (e.g. KPC) or metallo-carbapenemases (e.g. IMP, VIM and NDM), based on the hydrolytic mechanism at the active site. Ertapenem resistance in Enterobacter spp. is not uncommon and is mostly mediated by a combination of AmpC enzyme production and porin loss; imipenem and meropenem resistance is rarer, and more likely to be due to acquired carbapenemases. We report our experience with the first IMP metallo-b-lactamaseproducing Enterobacter cloacae isolated from patients admitted to Addenbrooke’s Hospital. In 2008–09, three isolates of meropenem-resistant E. cloacae were referred to the HPA Microbiology Services, Colindale, for confirmation. Two of three isolates were from blood cultures of haematology patients in an intensive care unit at the same time and one was from the urine of a renal transplant recipient. These isolates were resistant to gentamicin, tobramycin and tigecycline, and retained susceptibility only to amikacin and colistin. At the reference laboratory, the isolates were tested for carbapenem resistance by the BSAC agar dilution method and screened for carbapenemase production by the modified Hodge test. Carbapenemase genes were sought by PCR. PFGE of total DNA, digested with XbaI, was performed for strain comparison. All three meropenem-resistant E. cloacae isolates showed potentiation of the activity of imipenem in the presence of EDTA, indicating that they produced metallo-carbapenemases, and gave positive modified Hodge test results. PCR revealed the carbapenemases to be IMP types. A 651 bp intragenic fragment, from nucleotides 47 to 697 of the 741 bp blaIMP allele, was sequenced from all three isolates. The predicted amino acid sequences were identical to IMP-1 carbapenemase and, hence, different from other known IMP variants. PFGE showed that the three isolates belonged to a single strain; nevertheless, no epidemiological link was identified between the renal and haematology patients, or with hospitals elsewhere in the UK or overseas. This is the first identification of E. cloacae with an IMP carbapenemase in the UK based on submissions to HPA Microbiology Services, Colindale. It is part of a pattern whereby carbapenemase production is becoming more widespread in the Enterobacteriaceae, though, generally, KPC, NDM and VIM enzymes are more frequent than IMP types. – 6 The emergence of carbapenemases, which is often linked, as here, to resistance to multiple other drug classes, is a great public health concern. Producer strains should be actively sought to prevent their transmission among patients.