Development Of Carbapenem Resistance Research Articles

Emergence of diversity in carbapenemase-producing Escherichia coli ST131, England, January 2014 to June 2016.

Background Escherichia coli ST131, a global, high-risk clone, comprises fluoroquinolone resistance (FQ-R) mutations and CTX-M extended-spectrum beta-lactamases associated with the fimH30-encoding clades, C1 and C2. Further carbapenem resistance development in ST131 is a public health concern.AimThis observational study aimed to probe the diversity of carbapenemase-producing E. coli (CP E. coli) ST131 across England.MethodsST131 isolates were identified using whole-genome sequencing (WGS) data generated for all non-duplicate CP E. coli from human samples submitted to the national reference laboratory from January 2014 to June 2016. Antimicrobial resistance (AMR) gene content and single nucleotide polymorphism (SNP) data were compared against a published ST131 phylogeny and analysed alongside patient metadata.ResultsThirty-nine genetically diverse ST131 CP E. coli, from eight of nine regions, represented 10% of CP E. coli isolates sequenced. Ten and eight isolates were from the FQ-susceptible (FQ-S) clades A and B, while eight and 15 isolates belonged to the FQ-R clades C1 or C2, respectively. Seven distinct carbapenemases were identified: KPC-2 (21 isolates, 6 regions) frequently occurred among clade C2 isolates (n = 10). OXA-48-producers (10 isolates, 3 regions) were often from clade A (n = 5). NDM-1 (n = 4), NDM-5 (n = 1), VIM-1 (n = 1), VIM-4 (n = 1) and OXA-181 (n = 1) were also identified. Clade C2 isolates encoded more AMR genes than those from clades A (p = 0.02), B (p = 9.6 x 10−3) or C1 (p = 0.03).ConclusionWhen compared with its global predominance among ESBL-E. coli, ST131 represented a fraction of the CP E. coli received, belonging to diverse clades and encoding diverse carbapenemases. The greater accumulation of resistance genes in clade C2 isolates highlights the need for ongoing monitoring of this high-risk lineage.

Risk Factors for Development of Carbapenem Resistance Among Gram-Negative Rods.

BackgroundInfections due to carbapenem-resistant Gram-negative rods (CR-GNR) are increasing in frequency and result in high morbidity and mortality. Appropriate initial antibiotic therapy is necessary to reduce adverse consequences and shorten length of stay.MethodsTo determine risk factors for recovery on culture of CR-GNR, cases were retrospectively analyzed at a major academic hospital system from 2011 to 2016. Ertapenem resistance (ER-GNR) and antipseudomonal (nonertapenem) carbapenem resistance (ACR-GNR) patterns were analyzed separately. A total of 30951 GNR isolates from 12370 patients were analyzed, 563 of which were ER and 1307 of which were ACR.ResultsIn multivariate analysis, risk factors for ER-GNR were renal disease, admission from another health care facility, ventilation at any point before culture during the index hospitalization, receipt of any carbapenem in the prior 30 days, and receipt of any anti-methicillin-resistant Staphylococcus aureus (anti-MRSA) agent in the prior 30 days (c-statistic, 0.74). Risk factors for ACR-GNR were male sex, admission from another health care facility, ventilation at any point before culture during the index hospitalization, receipt of any carbapenem in the prior 30 days, and receipt of any anti-MRSA agent in the prior 30 days (c-statistic, 0.76).ConclusionsA straightforward scoring system derived from these models can be applied by providers to guide empiric antimicrobial therapy; it outperformed use of a standard hospital antibiogram in predicting infections with ER-GNR and ACR-GNR.

Contribution of acrB upregulation & OmpC/Ompk36 loss over the presence of blaNDM towards carbapenem resistance development among pathogenic Escherichia coli & Klebsiella spp.

Background & objectives:The global spread of carbapenem-resistant Enterobacteriaceae (CRE) is an emerging clinical problem. Hence, in this study, the plausible role of extended-spectrum beta-lactamases (ESBLs)/carbapenemases, OmpC/Ompk36, acrB and their combinations was explored among CRE.Methods:The minimum inhibitory concentration (MIC) of meropenem, enzyme-phenotypes (ESBLs/IR and metallo-beta-lactamase (MBL)/non-MBL carbapenemase), genotypes (blaTEM, blaSHV and blaCTX-M; blaNDM and blaVIM; blaKPC and blaOXA-48-like variants), acrB and outer membrane protein (OMP) expressions were analyzed with a total of 101 non-duplicate clinical isolates, obtained from various samples of patients visiting two tertiary care units of Eastern India during May 2013 - October 2016. This included Escherichia coli (n=36) and Klebsiella pneumoniae (n=65), categorized into two groups, namely Group I (resistant to all carbapenems; n=93; E. coli=34 and Klebsiella spp.=59) and Group II (non-resistant to all the carbapenems; n=8; E. coli=2 and Klebsiella spp.=6).Results:Though 88.17 per cent of Group I isolates exhibited ESBL property, the presence of carbapenemase activity (70.96%) and that of blaNDM gene (42/66: 63.63%) indicated their contributions towards the emergence of CRE. Further, porin loss and/or efflux pump activation among ESBL/carbapenemase-producing isolates heightened the MIC of meropenem from 64 to 256 mg/l (range exhibited by only ESBL/carbapenemase-producing isolates) to >256 mg/l.Interpretation & conclusions:These findings implied the major contribution of porin loss and/or efflux pump activation over the presence of ESBLs/carbapenemases in imparting carbapenem resistance in pathogenic bacteria.

Full title: Identification of potential drug targets against carbapenem resistant Enterobacteriaceae (CRE) strains using in silico gene network analysis

In the era where issue of antibiotic resistance is increasing at an alarming rate and resistance to most antibiotics has occurred, carbapenems have been the choice of drug against most infection because of its unique structure. However due to lack of antibiotic policy carbapenems too have achieved resistance and led to the emergence of carbapenem resistant Enterobacteriaceae (CRE). Members of Enterobacteriaceae family produce New Delhi metallo-beta-lactamase (NDM), a type of carbapenemase that can cleave carbapenems. Therefore, new anti-microbial targets need to be identified and for this purpose a holistic model is designed in this approach in order to find out the influential genes that are liable in imparting carbapenem resistance owing to the presence of NDM. In this research approach, firstly genes associated with resistance owing to NDM are collected from NCBI database. A total of 419 genes from E. coli that have been obtained from NCBI were subjected to STRING for protein-protein interaction analysis. Out of 419 genes, only 59 genes showed interaction. STRING gave 1679 functional partners at different confidence levels. In the next stage, clustering analysis of the 1679 functional partners was done using MCODE app of Cytoscape software which generated 71 clusters. Topological properties of the 804 functional partners obtained from the 71 clusters were done using Network Analyser plugin of Cytoscape software. 797 functional partners were streamlined and they were subjected to functional enrichment analysis by DAVID Database. This database finally gave 21 genes from 15 clusters, thus indicating that these 15 genes might have a crucial role in the development of carbapenem resistance owing to NDM. These genes can be targeted for designing new anti-microbial drugs in future by the way of gene silencing or altering the genetic expression.

Resistance to Carbapenems in Non-Typhoidal Salmonella enterica Serovars from Humans, Animals and Food.

Non-typhoidal serovars of Salmonella enterica (NTS) are a leading cause of food-borne disease in animals and humans worldwide. Like other zoonotic bacteria, NTS have the potential to act as reservoirs and vehicles for the transmission of antimicrobial drug resistance in different settings. Of particular concern is the resistance to critical “last resort” antimicrobials, such as carbapenems. In contrast to other Enterobacteriaceae (e.g., Klebsiella pneumoniae, Escherichia coli, and Enterobacter, which are major nosocomial pathogens affecting debilitated and immunocompromised patients), carbapenem resistance is still very rare in NTS. Nevertheless, it has already been detected in isolates recovered from humans, companion animals, livestock, wild animals, and food. Five carbapenemases with major clinical importance—namely KPC (Klebsiella pneumoniae carbapenemase) (class A), IMP (imipenemase), NDM (New Delhi metallo-β-lactamase), VIM (Verona integron-encoded metallo-β-lactamase) (class B), and OXA-48 (oxacillinase, class D)—have been reported in NTS. Carbapenem resistance due to the production of extended spectrum- or AmpC β-lactamases combined with porin loss has also been detected in NTS. Horizontal gene transfer of carbapenemase-encoding genes (which are frequently located on self-transferable plasmids), together with co- and cross-selective adaptations, could have been involved in the development of carbapenem resistance by NTS. Once acquired by a zoonotic bacterium, resistance can be transmitted from humans to animals and from animals to humans through the food chain. Continuous surveillance of resistance to these “last resort” antibiotics is required to establish possible links between reservoirs and to limit the bidirectional transfer of the encoding genes between S. enterica and other commensal or pathogenic bacteria.

Overproduction of efflux pumps caused reduced susceptibility to carbapenem under consecutive imipenem-selected stress in Acinetobacter baumannii.

PurposeAcinetobacter baumannii is an important pathogen in the nosocomial infections worldwide. Combining with carbapenemases, efflux pumps and outer membrane proteins (OMPs) have been thought to affect the development of carbapenem resistance in A. baumannii. This study aimed to investigate the contributions of different efflux pumps and OMPs in developing carbapenem resistance in a clinical isolate of A. baumannii and reveal the possible mechanism of overproduction of main efflux pumps.Patients and methodsIn this study, an imipenem-susceptible clinical isolate was identified as A. baumannii and named SZE. Several common carbapenemases were detected by polymerase chain reaction (PCR). Imipenem-selected mutants were selected from SZE by serial subcultivations on Mueller–Hinton agar, and the minimum inhibitory concentration (MIC) was detected. Gene expressions of four families of efflux pumps, five OMPs, and blaOXA-51 were determined by reverse transcription quantitative PCR, and comparisons were made between SZE strain and the imipenem-selected mutants. The adeRS system in SZE and its mutant was sequenced and aligned.ResultsUnder consecutive imipenem-selected stress, the MIC to imipenem increased gradually from 0.125 μg/mL to 8 μg/mL. The effect of resistance inducement was almost neutralized when treated with an efflux pump inhibitor. The expression of efflux pumps, adeB, adeG, and adeJ, was increased by 6.9-, 4.0-, and 2.1-fold in mutants, respectively, compared to SZE. A single mutation (G to A) at position 58 was detected in the regulatory adeRS system and possibly upregulated the adeB expression, and then affected the carbapenem resistance in A. baumannii strains.ConclusionIn conclusion, under consecutive imipenem-selected stress in vitro, A. baumannii strain evolved the ability to reduce susceptibility to a variety of antimicrobials by overproduction of efflux pumps. Especially, the resistance-nodulation-cell division super family and a nucleotide mutant in adeRS regulating system caused the overexpression of adeABC.

Insertion Sequence-Dependent <i>OmpK</i>36 Mutation Associated Ertapenem Resistance in Clinical <i>Klebsiella pneumoniae</i>

Extended-spectrum β-lactamases (ESBLs) and/or AmpC enzymes combined with deficiency of porins OmpK35 and OmpK36 are important for the development of carbapenem-resistant Klebsiella pneumoniae. We characterized the clinical K. pneumoniae human isolates and investigated the effect of meropenem induction on the ompK35 and ompK36 mutation to develop carbapenem resistance from six carbapenem-susceptible ESBL-producing K. pneumoniae strains. 163 clinical K. pneumoniae isolates were grouped mostly into the ESBL + AmpC (44.2%) and ESBL (42.9%) phenotypes. The resistance rate differed between cephalosporins (52.1% for cefepime - 97.5% for cefotaxime) and carbapenems (16% for meropenem - 28.2% for imipenem) (P blaTEM, blaSHV, blaCTX-M-3-like, and blaCTX-M-14-like of AmpA β-lactamase genes and blaDHA and blaCMY of AmpC β-lactamase genes. Compared to all 163 clinical isolates, the 56 carbapenem-resistant isolates carried less frequently of blaTEM, blaCTXM-14-like, and blaCTXM-3-like and more frequently of blaDHA-1 and blaCMY-2. The carbapenem-resistant isolates differed in prevalence against imipenem, ertapenem, and meropenem and lacked OmpK35 more frequently than OmpK36, but abnormal PCR amplicons were detected fewer in the Omp K35-deficient group than in the OmpK36-deficient group (32.5% vs. 68.4%, respectively). The carbapenem-resistant isolate mostly carried blaDHA (91.1%) and three isolates carried blaKPC-2. Following induction with meropenem insertion sequences in ompK36, not ompK36, were identified as IS5 for KP08, IS1 for KP15, and IS903 for KP16 isolates. OmpK36 deficiency increased resistance to ertapenem, but not imipenem and meropenem. Clinical isolates belonged mainly to ESBL + AmpC group and ESBL group with difference in resistance to cephalosporins and carbapenems, the bla genes. Carbapenem resistant isolates lacked OmpK35 expression, than the OmpK36 expression, Meropenem induction developed the carbapenem resistant isolates with insertion of different insertion sequences in ompK36, not ompK35.

Detection of Carbapenem resistance among Escherichia coli and Klebsiella pneumoniae isolates in a tertiary care hospital in South India

Introduction: Carbapenems are a class of ?lactam antibiotics with a broad spectrum of activity and greatest potency against Gram positive and Gram negative bacteria and their use has markedly increased in the past few decades. The development of carbapenem resistance has now become a grave concern in the treatment of diseases.Materials and Methods: The study was a laboratory based cross sectional study done with the main aim to detect the presence of carbapenem resistance among E. coli and K. pneumoniae isolates. A total of 550 samples (pus, urine, sputum, etc) were subjected to culture. The antibiotic susceptibility for various antibiotics was studied by Kirby Bauers Disk Diffusion Test. Carbapenem resistance and Metallo-betalacatamase (MBL) production was detected using Modified Hodge Test and EDTA Disk Synergy Test respectively.Result: A total of 129 isolates (43%) were meropenem resistant out of which 42 isolates (14%) were MBL producing. Carbapenase production was noted in 42.1% of E.coliand45.8% of K. pneumoniae. Twelve percent of the E. coli and 20.8% of the K. pneumoniae were found to have prevalence of MBL producers respectively.Conclusion: High incidence of carbapenem resistance and MBL producers was noted in the isolates. Given the emerging pattern of resistance to these third line antimicrobials and limited new molecules it is imperative to frame a new antibiotic policy and its strict implementation.

ST37 Klebsiella pneumoniae: development of carbapenem resistance in vivo during antimicrobial therapy in neonates.

To investigate the mechanism leading to in vivo carbapenem resistance development in Klebsiella pneumoniae. Carbapenemase was detected using the modified carbapenem inactivation method. β-lactamases resistant genes were identified by PCR and sequencing. Clonal relatedness was evaluated by random amplified polymorphic DNA and multiple locus sequence typing. The relationship between sequence typing and resistant genes was analyzed by using the chi-squared test. All ST37 carbapenem-resistant isolates were blaOXA-1 positive and all ST37 carbapenem-sensitive isolates were blaOXA-1 negative at Stage I. A significant relationship between carbapenem resistance and blaOXA-1 was observed. The blaOXA-1 -positive rate was significantly higher in ST37 K. pneumoniae than others. This is the first study about the development of carbapenem resistance in vivo potentially mediated by blaOXA-1 in ST37 K. pneumoniae among neonates.

Development of carbapenem resistance in Pseudomonas aeruginosa is associated with OprD polymorphisms, particularly the amino acid substitution at codon 170.

Pan-susceptible Pseudomonas aeruginosa (PSPA) clinical isolates carrying an OprD with loop 7 shortening (the group-1A allele) were found to rapidly develop carbapenem resistance under continuous selection pressure. We further studied whether OprD polymorphisms are associated with the potential to develop carbapenem resistance. OprD amino acid sequences of 126 PSPA clinical isolates were analysed to determine their STs using P. aeruginosa strain PAO1 as the control strain. Site-directed mutagenesis was performed in PAO1 to generate polymorphisms of interest. A disc diffusion method was used to select carbapenem-resistant variants from the mutant strains. Expression levels of oprD were determined by quantitative RT-PCR. MICs of carbapenems were determined by Etest. Forty-eight (38.1%) of the tested isolates carried the group-1A allele. Another two major STs, C1 and C2, both of which harboured an F170L polymorphism, were found in 21 (16.7%) and 39 (31.0%) isolates, respectively. The PAO1 type was also found in 14 (11.1%) isolates. Under continuous selective pressure, isolates of most STs developed carbapenem resistance at different numbers of passaging events; only those belonging to the PAO1 type remained susceptible. However, PAO1 mutants carrying either the oprD group-1A allele or the OprD-F170L polymorphism were able to develop carbapenem resistance. Reduced oprD expression triggered by continuous imipenem challenge was found in PAO1 mutants, but not in the PAO1 WT strain. OprD polymorphisms, particularly the F170L substitution and the specific shortening in loop 7, appear to determine the potential for P. aeruginosa to develop carbapenem resistance.

Assessment of Doripenem, Meropenem, and Imipenem against Respiratory Isolates of Pseudomonas aeroginosa in a Tertiary Care Hospital of North India.

Objective:Pseudomonas aeruginosa is one of the leading pathogen causing healthcare-associated infections, particularly in immunocompromised and critically ill patients. The development of carbapenem resistance in P. aeruginosa infections is worrisome. Data specifically comparing the susceptibility of the three available carbapenems are lacking in the Indian subcontinent.Materials and Methods:We evaluated the minimum inhibitory concentrations (MICs) of the three commonly used carbapenems– imipenem, meropenem, and doripenem against, 435 P. aeruginosa isolates obtained from respiratory samples and compared their susceptibility patterns to determine the best possible carbapenem among those available that may be used in combination regimes.Results:Overall, 222 (51.0%) of isolates were susceptible to doripenem followed by imipenem 206 (47.3%) and meropenem 195 (44.8%), respectively. Two hundred and sixty-two (60.23%) strains were intermediate or resistant to at least one carbapenem. The MIC90 of all three carbapenems was >32 μg/ml while the MIC50 of meropenem was 16 μg/ml which was higher than MIC50 of both imipenem (4 μg/ml) and doripenem (2 μg/ml).Conclusion:Our study revealed that doripenem exerted better in vitro activity against the tested bacteria compared to imipenem and meropenem, but the difference was not statistically significant.

Combinations of mutations in envZ, ftsI, mrdA, acrB and acrR can cause high-level carbapenem resistance in Escherichia coli.

The worldwide spread of ESBL-producing Enterobacteriaceae has led to an increased use of carbapenems, the group of β-lactams with the broadest spectrum of activity. Bacterial resistance to carbapenems is mainly due to acquired carbapenemases or a combination of ESBL production and reduced drug influx via loss of outer-membrane porins. Here, we have studied the development of carbapenem resistance in Escherichia coli in the absence of β-lactamases. We selected mutants with high-level carbapenem resistance through repeated serial passage in the presence of increasing concentrations of meropenem or ertapenem for ∼60 generations. Isolated clones were whole-genome sequenced, and the order in which the identified mutations arose was determined in the passaged populations. Key mutations were reconstructed, and bacterial growth rates of populations and isolated clones and resistance levels to 23 antibiotics were measured. High-level resistance to carbapenems resulted from a combination of downstream effects of envZ mutation and target mutations in AcrAB-TolC-mediated drug export, together with PBP genes [mrdA (PBP2) after meropenem exposure or ftsI (PBP3) after ertapenem exposure]. Our results show that antibiotic resistance evolution can occur via several parallel pathways and that new mechanisms may appear after the most common pathways (i.e. β-lactamases and loss of porins) have been eliminated. These findings suggest that strategies to target the most commonly observed resistance mechanisms might be hampered by the appearance of previously unknown parallel pathways to resistance.

Klebsiella pneumoniae: Development of Carbapenem Resistance due to Acquisition of bla NDM-1 During Antimicrobial Therapy in Twin Infants with Pneumonia.

Objectives: To identify the mechanism of in vivo development of carbapenem resistance in Klebsiella pneumoniae.Methods: Seven sequential isolates of K. pneumoniae were obtained from twin infants with pneumonia. Antimicrobial susceptibility testing was performed by agar dilution method. Carbapenemases including KPC and MβL were initially screened using phenotypic methods, and carbapenemase-encoding genes were identified by polymerase chain reaction and amplicon sequencing. Plasmids of all clinical isolates and the conjugants of resistant isolates were estimated by S1 pulsed-field gel electrophoresis (PFGE). Molecular typing were conducted by PFGE of XbaI-digested genomic DNA and multilocus sequence typing.Results: For old brother, the first and third isolates were susceptible to meropenem, whereas the second and fourth isolates were resistant (MICs 16 mg/L). The first and second isolates from the young brother were susceptible to meropenem whereas the third isolate was resistant. All the resistant isolates produced NDM-1 metallo-β-lactamase. PFGE of XbaI-digested DNA revealed almost identical patterns with similarity indices of above 92% for all the seven isolates. All the isolates had the same sequence type named sequence type 37 (ST37).Conclusion: To our knowledge, this is the first documented case of development of carbapenem resistance in vivo mediated by NDM-1 metallo-β-lactamase in K. pneumoniae during treatment of pneumonia with meropenem.

A de-escalation protocol for febrile neutropenia cases and its impact on carbapenem resistance: A retrospective, quasi-experimental single-center study.

Our objective was to evaluate the impact of using an imipenem de-escalation protocol for empiric febrile neutropenia on the development of carbapenem resistance. A pre-post intervention design was used. The intervention was adopting the imipenem de-escalation approach, which began on January 1, 2012. A retrospective chart review of cases of febrile neutropenia bacteremia was performed one year before and one year after the intervention. We compared the development of carbapenem resistance between the two study periods. Seventy-five episodes of febrile neutropenia bacteremia were included in the study. They had similar demographics, clinical features and outcomes. There were 78 and 12 pathogens in the primary and follow-up blood cultures, respectively. Approximately 61% and 66% of the primary and follow-up blood cultures, respectively, were gram-negative bacteria with similar carbapenem resistance profiles in the two study periods. In our study population, 57% of the gram-negative bacteria were ESBL pathogens. The resistance of the gram-negative bacteria to piperacillin/tazobactam (72% versus 53%, p=0.161), imipenem (16% versus 11%, p=0.684), and meropenem (8% versus 16%, p=0.638) did not significantly change after our policy change. In conclusion, the use of the carbapenem de-escalation approach for febrile neutropenia in our institution was not associated with an increase in carbepenem resistance. Future prospective multi-center studies are recommended to further confirm the current findings.

Mechanisms of Carbapenem Resistance in Multidrug-Resistant Clinical Isolates of Pseudomonas aeruginosa from a Croatian Hospital.

Pseudomonas aeruginosa is an opportunistic pathogen, one of the leading causes of nosocomial infections such as pneumonia, urinary tract infections, and bacteraemia. The bacterial resistance to structurally unrelated antibiotics and its spread within hospitals limits the efficient antimicrobial options and patients' outcome. Carbapenems are important agents for the therapy of infections due to multidrug-resistant (MDR) P. aeruginosa; hence, the development of carbapenem resistance severely hampers effective therapeutic options. The aim of this investigation was to examine mechanisms of carbapenem resistance and genomic diversity in carbapenem-resistant MDR strains of P. aeruginosa, which caused an outbreak among patients in Clinical Hospital Rijeka. Most of the isolates showed decreased expression of porin that is important for the entry of carbapenems (oprD). Overexpression of MexAB-OprM, MexCD-OprJ, and MexEF-OprN efflux systems was observed in many of the isolates. Production of metallo-β-lactamases was not detected. Typing by pulsed-field gel electrophoresis discriminated the isolates into five clusters. The clonal distribution of the strains was related to the location of hospital departments where the isolates were collected, which implies that most of the infections were caused by spread of the epidemic strains within the hospital.

Carbapenemase-ProducingKlebsiella pneumoniaein Hospital, Singapore, 2011

Carbapenemase-Producing<i>Klebsiella pneumoniae</i>in Hospital, Singapore, 2011

Development of carbapenem resistance during therapy for non-typhoid Salmonella infection

Multidrug-resistant Salmonella infection is a global problem, and carbapenems may represent the last therapeutic choice. We report a case of infection caused by ceftriaxone-resistant and ciprofloxacin-resistant Salmonella enterica serotype Typhimurium. A blaCMY-2-containing Tn6092, located on a self-transferable IncI1 plasmid, was found in all isolates derived from the patient. During ertapenem treatment, the strain developed carbapenem resistance. Apart from the OmpD deficiency found in all isolates, the strain further developed OmpC deficiency through a single gene mutation, and became carbapenem-resistant. Salmonella appears to be very plastic in developing antimicrobial resistance. Care must be taken by physicians when treating multidrug-resistant Salmonella infection.

Molecular diversity in mechanisms of carbapenem resistance in paediatric Enterobacteriaceae

Development of carbapenem resistance in Enterobacteriaceae has impacted Clinical and Laboratory Standards Institute (CLSI) guidelines, infection control approaches and treatment strategies. The clinical, phenotypic and genotypic characteristics of carbapenem-resistant Enterobacteriaceae (CRE) infections at paediatric referral centres are not well described. CRE were identified through the clinical microbiology laboratory at Seattle Children's Hospital (Seattle, WA). Clinical data were retrieved from medical records. Resistance testing, polymerase chain reaction (PCR) for resistance determinants, and Escherichia coli transformation were carried out for each isolate. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were used to characterise strain relatedness. PCR amplification and sequencing as well as sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) were used to investigate porin alterations. Six CRE isolates were identified between 2002 and 2010. Significant molecular diversity was documented in their mechanisms of resistance, including plasmid-mediated serine carbapenemase (KPC) and metallo-β-lactamase (IMP), chromosomally encoded β-lactamase (SME) and porin alterations with extended-spectrum β-lactamases. Patients had underlying health conditions and were from geographically diverse regions. In one case, PFGE of serial isolates documented the development of resistance in a previously susceptible strain. Molecular investigation of this strain identified insertion of the genetic mobile element insertion sequence IS Ecp1 in the ompK36 gene, conferring a functional porin alteration as demonstrated by SDS-PAGE. This is the first description of porin disruption by IS Ecp1 in a CTX-M-15-positive isolate. This is the largest report of paediatric CRE to date. This diverse description of demographic, phenotypic and molecular characteristics highlights the challenge of CRE infections in high-risk paediatric patients and that attention to emerging resistance mechanisms (including membrane alteration) at paediatric referral centres is essential.

The emergence of carbapenem resistance in Enterobacteriaceae in south africa

The high levels of extended-spectrum s-lactamase (ESBL) production recorded among invasive Enterobacteriaceae in South Africa, especially documented for Klebsiella pneumoniae, in both the public sector (55-74%), as reported by Bamford et al in this edition of the SAJEI, and in the private sector (55-60%) shown previously, is disconcerting. 1,2 Carbapenems are the cornerstone of therapy for patients with serious infections caused by ESBL-producing organisms. The high ESBL prevalence amongst bacteraemic pathogens places a tremendous strain on the use of these agents as directed therapy, and empirically as well. In the private sector, the use of imipenem, meropenem and ertapenem, more than doubled in terms of monthly units sold, between January 2009 and June 2011. Not only is this consumption, through selective pressure, creating the ideal environment for the development of carbapenem resistance among the enterobactericeae, but at the same time, carbapenem use has been shown to be a risk factor for subsequent infections with ESBL-producing organisms. 3 Excessive use, including inappropriate duration of therapy, is therefore selecting for the very resistance that the class is being used for. A vicious cycle is established in the process. Therefore, the emergence of carbapenem resistance among Enterobacteriaceae in South Africa is inevitable, and as documented for the public sector in this edition of the SAJEI, of major concern.

Antibiotic susceptibility profiles of European Bacteroides fragilis with reduced carbapenem susceptibility

Sir, Bacteroides fragilis usually causes infection together with other aerobic and anaerobic bacteria and is one of the most important anaerobic pathogens responsible for a variety of significant clinical infections including intra-abdominal infections, complicated skin and skin structure infections, bacteraemia and deep abscesses. Antibiotic resistance in this important pathogen has been noted and is increasing in many parts of the world. The preferred agents of choice for B. fragilis infections include broad-spectrum penicillins, metronidazole, clindamycin, piperacillin/tazobactam, carbapenems, moxifloxacin and, more recently, tigecycline. Tigecycline was recently shown to exhibit promising activity against European isolates of various anaerobes, including Bacteroides spp. Today the majority of anaerobic infections are treated empirically and minimal, if any, antimicrobial susceptibility testing is performed. With the knowledge that antimicrobial resistance is increasing to many classes of antimicrobial agents used to treat anaerobic infections, active ongoing surveillance of resistance patterns of anaerobes is advantageous. The use of carbapenems as one of the preferred agents of choice has ultimately led to the development of carbapenem resistance and the recognition of the metallo-b-lactamase gene cfiA and its expression. This report documents the in vitro activity of six antimicrobial agents against B. fragilis isolates collected in Europe during 2007–08. All isolates were derived from complicated intra-abdominal infections, gastrointestinal, wounds, blood and other body fluid sources. Isolates were identified to genus and species by the local laboratory and confirmed at a reference laboratory. Only one isolate per patient was accepted. There were 928 clinical isolates of B. fragilis collected between 2007 and 2008 from 20 unique laboratory sites in six European countries (Belgium, the Czech Republic, France, Germany, the UK and Hungary). All isolates were sent to a central laboratory in the USA (Laboratories International for Microbiology Studies, a subsidiary of International Health Management Associates, Inc., Schaumburg, IL, USA) for confirmation of identification and susceptibility testing. MICs were determined by agar dilution as specified by the CLSI. The following antimicrobial agents were tested with their dilution ranges (expressed in mg/L): tigecycline (0.06–32); clindamycin (0.25–8); metronidazole (0.12–16); piperacillin/tazobactam (0.06/4–64/4); meropenem (0.06–8); and cefoxitin (2–32). MIC interpretive criteria followed published breakpoints established by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) where applicable. Since no EUCAST guidelines were available for cefoxitin, CLSI breakpoints were used. FDA anaerobe breakpoints were used for tigecycline. Quality control testing was done following CLSI guidelines. Table 1 lists the susceptibilities of B. fragilis clinical isolates to the six antimicrobial agents. Isolates were generally susceptible to the antimicrobial agents with percentage susceptibilities of 87% with the exception of clindamycin for which 22.6% of isolates were resistant. The most active agents by MIC90 were meropenem (MIC90 0.5 mg/L), metronidazole (MIC90 1 mg/L) and piperacillin/tazobactam and tigecycline (MIC90 2 mg/L) Research letters