Chromosomal location of blaCTX-M genes in clinical isolates of Escherichia coli from Germany,The Netherlands and the UK

This study aimed to detect and characterise clinical Escherichia coli isolates suspected of carrying chromosomally encoded CTX-M enzymes. Escherichia coli (n = 356) obtained in Germany, The Netherlands and the UK (2005–2009) and resistant to third-generation cephalosporins were analysed for the presence of ESBL-/AmpC-encoding genes within the European SAFEFOODERA-ESBL project. β-Lactamases and their association with IS26 and ISEcp1 were investigated by PCR. Isolates were typed by phylogenetic grouping, MLST and PFGE. Plasmids were visualised by S1 nuclease PFGE, and the location of bla_CTX-M genes was determined by Southern hybridisation of XbaI-, S1- and I-CeuI-digested DNA. ESBL enzymes could not be located on plasmids in 17/356 isolates (4.8%). These 17 isolates, from different countries and years, were ascribed to phylogenetic groups D (9), B2 (6) and B1 (2), and to seven sequence types, with ST38 being the most frequent (7 phylogroup D isolates). Eleven isolates produced CTX-M-15. bla_CTX-M-15 genes were associated with ISEcp1. The remaining isolates expressed the CTX-M group 9 β-lactamases CTX-M-14 (4), CTX-M-9 (1) and CTX-M-51 (1). bla_CTX-M probes hybridised with I-CeuI- and/or XbaI-digested DNA, but not with S1-digested DNA, corroborating their chromosomal location. To summarise, only 4.8% of a large collection of ESBL-producing E. coli isolates harboured chromosomal bla_CTX-M genes. These isolates were of human origin and belonged predominantly to ST38 and ST131, which possibly indicates the role of these sequence types in this phenomenon. However, heterogeneity among isolates was found, suggesting that their spread is not only due to the dispersion of successful E. coli clones.     

High prevalence of CTX-M-15-producing Klebsiella pneumoniae isolates in Asian countries: diverse clones and clonal dissemination

The characteristics of 218 Klebsiella pneumoniae isolates from patients with hospital-acquired pneumonia in nine Asian countries were investigated. In total, 92 isolates (42.2%) produced extended-spectrum β-lactamases (ESBLs), amongst which 67 (72.8%) possessed CTX-M ESBL genes; CTX-M-15 was the major ESBL (55 isolates; 59.8%). Multilocus sequence typing (MLST) and plasmid replicon typing were performed to investigate the genetic backgrounds of the 55 CTX-M-15-producing K. pneumoniae isolates. Twenty-five sequence types (STs) were identified. Clonal complex 11 (CC11) including ST11 was the most prevalent clone (20 isolates; 36.4%) and was distributed in all Asian countries except Taiwan. ST15 was the next most frequently identified clone (8 isolates; 14.5%). An IncFIIA-type plasmid was predominantly associated with bla_CTX-M-15 (45 isolates; 81.8%). However, another plasmid type (IncA/C) was also identified and replicon types of seven isolates could not be determined. The high prevalence of CTX-M-15 amongst K. pneumoniae isolates in Asian countries may be due both to the acquisition of plasmids carrying bla_CTX-M-15 and the spread of certain clones such as ST11 and ST15.     

Seven-year surveillance of the prevalence of antimicrobial-resistant Escherichia coli isolates,with a focus on ST131 clones,among healthy people in Osaka,Japan

ObjectivesEscherichia coli (E. coli) is an indicator of antimicrobial resistance, and some strains of E. coli cause infectious diseases. E. coli sequence type 131 (ST131) – a global antimicrobial-resistant pandemic E. coli clone – is frequently detected in clinical specimens. Antimicrobial-resistant bacteria are monitored via national surveillance in clinical settings; however, monitoring information in non-clinical settings is limited. This study elucidated antimicrobial resistance trends of E. coli and dissemination of ST131 among healthy people in non-clinical settings.MethodsThis study collected 517 E. coli isolates from healthy people in Osaka, Japan, between 2013 and 2019. It analysed antimicrobial susceptibility of the isolates and detected the bla and mcr genes in ampicillin-resistant and colistin-resistant isolates, respectively, and the ST131 clone.ResultsAntimicrobial resistance rates of the bacteria isolated from healthy people in non-clinical settings were lower than for those in clinical settings. The resistance of the isolates to cefotaxime (4.4%) and ciprofloxacin (13.5%) gradually increased during the study period. In 23 cefotaxime-resistant isolates, the most frequent bla genes belonged to the bla_CTX-M-9 group, followed by bla_CTX-M-1 goup, bla_TEM and bla_CMY-2. One mcr-1-harbouring colistin-resistant isolate was detected in 2016. The incidence of the E. coli O25b-ST131 clone was approximately 5% until 2015 and 10% after 2016.ConclusionBoth ciprofloxacin resistance and O25b-ST131 clone frequency increased during the study period. Antimicrobial-resistant bacteria gradually spread in healthy people in non-clinical settings; one reason behind this spread was dissemination of global antimicrobial-resistant pandemic clones.     

In vitro activity of rifaximin against clinical isolates of Escherichia coli and other enteropathogenic bacteria isolated from travellers returning to the UK

Rifaximin is licensed in the EU and USA for treating travellers’ diarrhoea caused by non-invasive bacteria. Selection for resistance mechanisms of public health significance might occur if these are linked to rifamycin resistance. Rifaximin MICs were determined by agar dilution for 90 isolates each of Escherichia coli, Shigella spp., nontyphoidal Salmonella enterica, typhoidal S. enterica and Campylobacter spp., an additional 60 E. coli with CTX-M ESBLs isolated from patients with travellers’ diarrhoea, and 30 non-diarrhoeal carbapenemase-producing E. coli. Comparators were rifampicin, ciprofloxacin, azithromycin, trimethoprim/sulfamethoxazole and doxycycline. Isolates with rifaximin MICs>32 mg/L were screened for arr genes, and critical rpoB regions were sequenced. Rifaximin was active at ≤32 mg/L against 436/450 (96.9%) diverse Enterobacteriaceae, whereas 81/90 (90%) Campylobacter spp. were resistant to rifaximin at ≥128 mg/L. Rifaximin MICs were ≥128 mg/L for two Shigella and five MDR E. coli producing NDM (n = 3), OXA-48 (n = 1) or CTX-M-15 (n = 1). Two of the five MDR E. coli had plasmids harbouring arr-2 together with bla_NDM, and two (one each with bla_NDM and bla_CTX-M-15) had His526Asn substitutions in RpoB. The rifamycin resistance mechanism remained undefined in one MDR E. coli isolate (with bla_OXA-48) and the two Shigella isolates. Rifaximin showed good in vitro activity against diverse Enterobacteriaceae but was largely inactive against Campylobacter spp. Rifaximin has potential to co-select MDR E. coli in the gut flora, but much stronger associations were seen between ESBL and/or carbapenemase production and resistance to alternative treatments for travellers’ diarrhoea, notably ciprofloxacin and azithromycin.     

Genetic and structural insights into plasmid-mediated extended-spectrum β-lactamase activity of CTX-M and SHV variants among pathogenic Enterobacteriaceae infecting Indian patients

Resistance to third-generation cephalosporins (3GCs) mediated by extended-spectrum β-lactamases (ESBLs) in pathogenic Enterobacteriaceae is considered a major public health threat in India. This study deals with the detection of plasmid-mediated bla_CTX-M, bla_SHV and bla_OXA genes, understanding their contribution to the ESBL phenotype, and their molecular interaction with 3GCs. More than 87% of isolates showed 3GC resistance, with ESBL production in 60.0% of Escherichia coli and 47.7% of Klebsiella pneumoniae. Molecular characterisation revealed the presence of bla_CTX-M-15 (29.8%), bla_{CTX-M-truncated} (1.3%), bla_CTX-M-27 (0.7%), bla_SHV-1 (20.5%), bla_SHV-11 (2.0%), bla_SHV-42 (0.7%) and bla_OXA-1 (9.9%), among which bla_CTX-M variants and bla_SHV-42 were ESBLs. Phylogenetic analysis predicted strong selection pressure on all bla_CTX-M variants, bla_SHV-11 and bla_SHV-42. The instability index and Gibbs free folding energy change (ΔΔG) predicted decreased stability of SHV-11 and SHV-42. Mutations of CTX-M-truncated, SHV-11 and SHV-42 located in the core region of the enzymes were found to be functional/pathogenic in nature. The catalytic pockets of CTX-M-15 and SHV-42 had the greatest molecular surface area, which might explain their expanded substrate spectrum towards oxyimino-cephalosporins. Molecular dynamics analysis indicated different structural flexibility of CTX-M-truncated compared with the other enzymes. Amino acid alterations resulted in a change of orientation of catalytic residues of class A β-lactamases that might affect their catalytic processes. Molecular interactions revealed higher catalytic efficiency (ΔG and K_m) of CTX-M-15, CTX-M-truncated, CTX-M-27, SHV-11 and SHV-42 compared with their respective wild-types. This study provides useful insights into ESBL production of pathogenic Enterobacteriaceae in India that might help in the development of new antibiotics.     

Antibiotic resistance genes in phage particles isolated from human faeces and induced from clinical bacterial isolates

Phage particles have emerged as elements with the potential to mobilise antibiotic resistance genes (ARGs) in different environments, including the intestinal habitat. This study aimed to determine the occurrence of ARGs in phage particles present in faecal matter and induced from strains isolated from faeces. Nine ARGs (bla_TEM, bla_{CTX-M-1 group}, bla_{CTX-M-9 group}, bla_OXA-48, qnrA, qnrS, mecA, sul1 and armA) were quantified by qPCR in the phage DNA fractions of 150 faecal samples obtained from healthy individuals who had not received antibiotic treatment or travelled abroad in the 3 months prior to sample collection. On the suspicion that the detected particles originated from bacterial flora, 82 Escherichia coli and Klebsiella pneumoniae isolates possessing at least one identified ARG (bla_TEM, bla_{CTX-M-1 group}, bla_{CTX-M-9 group}, armA, qnrA, qnrS and sul1) were isolated and their capacity to produce phage particles carrying these ARGs following induction was evaluated. Of 150 samples, 72.7% were positive for at least one ARG, with bla_TEM and bla_{CTX-M-9 group} being the most prevalent and abundant. Of the 82 isolates, 51 (62%) showed an increase in the number of copies of the respective ARG in the phage fraction following induction, with bla_TEM, bla_{CTX-M-1 group}, bla_{CTX-M-9 group} and sul1 being the most abundant. Phages induced from the isolates were further purified and visualised using microscopy and their DNA showed ARG levels of up to 10¹⁰ gene copies/mL. This study highlights the abundance of phage particles harbouring ARGs and indicates that bacterial strains in the intestinal habitat could be source of these particles.     

High-level and novel mechanisms of carbapenem resistance in Gram-negative bacteria from tertiary hospitals in Nigeria

To determine the occurrence and molecular basis of carbapenem resistance in Gram-negative bacteria from tertiary hospitals in Nigeria, 182 non-duplicate Gram-negative bacterial isolates were investigated for antimicrobial susceptibility, presence of carbapenemases (tested phenotypically and genotypically), random amplified polymorphic DNA (RAPD) typing, plasmid sizing and replicon typing. Minimum inhibitory concentrations of carbapenems showed a high degree of resistance, with 67 isolates (36.8%) being resistant to all carbapenems, of which 40 (59.7%) produced enzymes able to hydrolyse imipenem. PCR and sequencing identified only 10 isolates (5.5%) carrying known carbapenemase genes, including bla_NDM, bla_VIM and bla_GES. The majority of phenotypically carbapenem-resistant and carbapenemase-producing isolates did not carry a known carbapenemase gene. Transconjugant or transformant plasmid sizes were estimated to be 115 kb for bla_NDM- and 93 kb for bla_VIM-carrying plasmids. These plasmids were untypeable for replicon/incompatibility and transferred various other genes including plasmid-mediated quinolone resistance (PMQR) genes and bla_CTX-M-15. Typing showed that the isolates in this study were not clonally related. There is a high level of carbapenem resistance in Nigeria. As well as the globally relevant carbapenemases (bla_NDM, bla_VIM and bla_GES), there are other unknown gene(s) or variant(s) in circulation able to hydrolyse carbapenems and confer high-level resistance.     

Role of rifampicin in limiting Escherichia coli O157:H7 Shiga-like toxin expression and enhancement of survival of infected BALB/c mice

Plasmid-mediated 16S rRNA methylases such as ArmA, which confer high levels of resistance to aminoglycosides, are increasingly reported in Enterobacteriaceae. This study investigated the molecular mechanism of β-lactam and aminoglycoside resistance in extended-spectrum β-lactamase (ESBL)-producing Salmonella enterica serotype Infantis isolated at the 53-bed neonatology ward of University Hospital Benabib in Constantine, Algeria. From September 2008 to January 2009, 200 S. enterica isolates were obtained from 138 patients (age range 8-80 months) hospitalised in the neonatology ward. Most isolates were from stool cultures, but also from two blood cultures and one gastric fluid. The isolates were multidrug-resistant and produced TEM-1 and CTX-M-15 enzymes as well as the 16S RNA methylase ArmA. The armA, bla_CTX-M-15 and bla_TEM-1 genes were located on the same 140-kb self-transferable plasmid belonging to the IncL/M incompatibility group. All of the S. Infantis isolates belonged to a single clone. Increased infection control measures and thorough biodecontamination of the rooms led to control of the outbreak but did not eradicate the epidemic strain. This study further illustrates the global emergence of ArmA methylase and its frequent association with bla_CTX-M genes. Spread of 16S RNA methylase determinants at the same level as bla_CTX-M genes in Enterobacteriaceae may seriously compromise the efficacy of aminoglycosides for treating Gram-negative infections.     

Molecular analysis of multiresistant porcine Salmonella enterica subsp. enterica serovar Bredeney isolates from Southern Brazil: identification of resistance genes, integrons and a group II intron

The relationships of 83 porcine Salmonella enterica subsp. enterica serovar Bredeney isolates obtained at two slaughterhouses in Southern Brazil were analysed by XbaI and BlnI macrorestriction analysis, plasmid profiling and determination of antimicrobial resistance patterns. Twenty-nine XbaI and 30 BlnI macrorestriction patterns were identified. The 72 plasmid-bearing isolates exhibited 20 different plasmid profiles. Multiresistance was detected in 49 isolates (59%), of which 39 isolates showed at least resistance to sulfonamides, tetracyclines, chloramphenicol, streptomycin, kanamycin and/or ampicillin. A representative subset of 12 isolates was chosen for identification of resistance genes, their localisation and transferability. The sulfonamide resistance genes sul1, sul2 and sul3, the tetracycline resistance genes tet(A) and tet(B), the phenicol resistance genes catA1 and floR, the streptomycin resistance gene strA, the kanamycin resistance gene aphA1 and the ampicillin resistance gene bla_TEM were detected and found to be located most frequently on plasmids. In addition, class 1 and 2 integrons with the cassette arrangements dfrA21/bla_OXA-129/aadA1 and dfrA1/sat1/aadA1, respectively, were detected. A group II intron was found to be inserted into the 59-base element of an aadA1 gene cassette in a class 1 integron. This study revealed a wide genomic variety among the S. Bredeney isolates, and the high number of multiresistant isolates may point towards the risks that these S. Bredeney isolates can represent to human health.     

Characterisation of a cointegrate plasmid harbouring blaNDM-1 in a clinical Salmonella Lomita strain

This study aimed to characterise the molecular events underlying formation and evolution of a cointegrate plasmid harbouring bla_NDM-1 and bla_CMY-2 in a clinical Salmonella Lomita (S. Lomita) strain. The Salmonella strain SL131 was found to harbour two multidrug resistant (MDR) plasmids. One plasmid, pSL131_IncHI2, is a typical IncHI2 plasmid containing bla_OXA-1, catB3, arr-3, sul1, qnrB4 and bla_DHA-1 in a complex class 1 integron. The other plasmid, pSL131_IncA/C-IncX3, is a bla_NDM-1-bearing cointegrate plasmid consisting of IncX3 and IncA/C backbones, the formation of which is mediated by IS26. Stability assay showed that the cointegrate plasmid was highly stable in its natural host – S. Lomita – but would readily resolve into single plasmids upon conjugation, during which the IncX3 bla_NDM-1-bearing plasmid could be transferred to Escherichia coli strain EC600. Plasmid evolution through integration of two or more MDR plasmids would not only expand the resistance profile of the resultant plasmid, but also broaden the host spectrum of such resistance-encoding mobile elements. Better understanding of the underlying and triggering mechanisms of cointegration may facilitate development of intervention measures to curb formation and dissemination of such elements.     

Faropenem resistance causes in vitro cross-resistance to carbapenems in ESBL-producing Escherichia coli

ObjectiveFaropenem is an oral penem drug with activity against Gram-positive and Gram-negative bacteria, including CTX-M-15-type extended spectrum beta-lactamase (ESBL)-producing Enterobacteriales and anaerobic bacteria. As there are structural similarities, there is concern for the development of carbapenem cross-resistance; however, there are no studies confirming this. This study examined whether in vitro development of faropenem resistance in Escherichia coli isolates would result in cross-resistance to carbapenems.MethodsFour well-characterized E. coli isolates from the US Centers for Disease Control and Prevention antibiotic resistance isolate bank were utilized. Three isolates (NSF1, NSF2 and NSF3) are ESBL producers (CTX-M-15) and one (NSF4) is pan-susceptible. Faropenem minimum inhibitory concentrations (MICs) were determined and resistance was induced by serial passaging in increasing concentrations of faropenem. Susceptibility to carbapenems was determined and whole-genome sequencing (WGS) was performed to identify the underlying genetic mechanism leading to carbapenem resistance.ResultsFaropenem MIC increased from 1 mg/L to 64 mg/L within 10 days for NSF2 and NSF4 isolates, and from 2 mg/L to 64 mg/L within 7 days for NSF1 and NSF3 isolates. Reduced carbapenem susceptibility (ertapenem MIC ≥8 mg/L, doripenem/meropenem ≥2 mg/L and imipenem ≥1 mg/L) developed among three CTX-M-15-producing isolates that were faropenem-resistant, but not in NSF4 isolate that lacked ESBL enzyme. WGS analysis revealed non-synonymous changes in the ompC gene among three CTX-M-15-producing isolates, and a single nucleotide polymorphism (SNP) in the envZ gene in NSF4 isolate.ConclusionInduced resistance to faropenem causes cross-resistance to carbapenems among E. coli isolates containing CTX-M-15-type ESBL enzymes.     

Role of IncHI2 plasmids harbouring blaVIM-1, blaCTX-M-9, aac(6')-Ib and qnrA genes in the spread of multiresistant Enterobacter cloacae and Klebsiella pneumoniae strains in different units at Hospital Vall d'Hebron, Barcelona, Spain

Seven Enterobacter cloacae isolates and seven Klebsiella pneumoniae isolates harbouring a phenotype compatible with the production of a metallo-β-lactamase were recovered between 2009 and 2011 in three Intensive Care Units of Hospital Vall d'Hebron (Barcelona, Spain). The presence of bla(VIM), bla(IMP), bla(NDM), bla(CTX-M), aac(6')-Ib, qnrA, qnrB and qnrS genes was screened by polymerase chain reaction (PCR) and sequencing. Clonal relatedness of the isolates was assessed by pulsed-field gel electrophoresis (PFGE) and, in the case of K. pneumoniae isolates, by multilocus sequence typing (MLST). PCR-based replicon typing, Southern hybridisation, plasmid double-locus sequence typing and MOB relaxase classification methods were used to identify and characterise the plasmids carrying the resistance genes. Transferability of the identified plasmids was tested by conjugation assays. All 14 isolates were found to carry bla(VIM-1), bla(CTX-M-9) (except one isolate), aac(6')-Ib and qnrA genes. Clonality assessment demonstrated that E. cloacae isolates were distributed in three clonal clusters, whereas all of the K. pneumoniae isolates belonged to one unique clone, identified as sequence type ST252. All studied isolates harboured a large conjugative IncHI2 MOB(H11) plasmid carrying all of the detected resistance genes. Plasmid DNA analysis showed that all of them belonged to the ST1 IncHI2 plasmid cluster and shared the same relaxase partial sequence. In conclusion, the present study describes the dissemination within a hospital of multiresistant E. cloacae and K. pneumoniae isolates producing VIM-1. A complex clonal epidemiology of the E. cloacae isolates was observed and plasmid DNA analysis strongly supports horizontal exchanges of the same IncHI2 plasmid between different strains and species.     

The prevalence of colistin resistance in Escherichia coli and Klebsiella pneumoniae isolated from food animals in China: coexistence of mcr-1 and blaNDM with low fitness cost

Increasing colistin resistance is a global concern because colistin is used as a last resort for the treatment of carbapenem-resistant Enterobacteriaceae infections. The plasmid-mediated colistin resistance gene, mcr-1 was found in distinct bacterial species isolated from humans, animals, and the environment. In this study, farms in four different agricultural provinces in China were investigated to determine the occurrence of the antimicrobial resistance and related genes. A total of 373 Escherichia coli and 54 Klebsiella pneumoniae were isolated from 510 non-duplicated samples. Of the E. coli and K. pneumoniae isolates, 72.7% and 66.7%, respectively, were susceptible to colistin. Isolates resistant to colistin comprised 46.6% of the samples isolated from Shandong, and 17.8% and 16.4% of the samples from Jilin and Henan, respectively. Twenty-six carbapenem-resistant E. coli isolates were resistant to colistin, in which both mcr-1 and bla_NDM were present. Specifically, the co-existence was found in isolates from animals and sewage. Most of the resistance genes were located on plasmids and were 40–244 kilobases. Growth curves of transconjugants carrying mcr-1, bla_NDM-1, bla_NDM-4, bla_NDM-5, and bla_NDM-9 showed a low fitness cost compared with the recipient. In conclusion, mcr-1 was widespread in E. coli and K. pneumoniae isolated from farms in China. Co-existence of mcr-1 and bla_NDM-9 was identified in different sequence types of E. coli with low fitness cost from various origins, indicating an urgent need to take measures for decreasing dissemination.     

Hospital sewage water: a reservoir for variants of New Delhi metallo-β-lactamase (NDM)- and extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae

New Delhi metallo-β-lactamase (NDM)-producing Enterobacteriaceae have become a threat to public health. Hospital sewage is generally unexplored, having the potential to harbour bacteria causing healthcare-associated infections. Hence, this study was initiated to monitor NDM-producing Enterobacteriaceae in hospital sewage water. A total of 32 isolates with bla_NDM variants were detected in hospital sewage water, including 17 Escherichia coli, 8 Citrobacter freundii, 4 Shigella boydii, 2 Citrobacter braakii and 1 Citrobacter farmeri, showing resistance to all antibiotics except colistin. All 32 isolates carried bla_NDM (9 bla_NDM-1, 11 bla_NDM-4, 10 bla_NDM-5 and 2 bla_NDM-7), 24 isolates carried bla_CMY variants (1 bla_CMY-2, 3 bla_CMY-4, 5 bla_CMY-6, 11 bla_CMY-42, 2 bla_CMY-86 and 2 bla_CMY-139), 20 isolates carried bla_OXA-type (17 bla_OXA-1 and 3 bla_OXA-48), 19 isolates carried bla_CTX-M and 9 isolates carried ampC on conjugative plasmids of IncFIA, IncFIB, IncFIC, IncP, IncY, IncHI1 and IncI1 types. In E. coli, coexistence of bla_NDM-1 with bla_CMY-6 and bla_CMY-139, of bla_NDM-4 with bla_CMY-6, bla_CMY-42 and bla_CMY-86, of bla_NDM-5 with bla_CMY-6 and bla_CMY-42, and of bla_NDM-7 with bla_CMY-6 was observed. NDM-5-producing S. boydii and NDM-7-producing C. freundii were identified as well as detection of an association of bla_NDM-4 and bla_OXA-48 in C. braakii and C. farmeri. A class 1 integron was also found on a plasmid. ISAba125 and bleomycin genes were found surrounding all bla_NDM variants. The emergence and dissemination of bla_NDM variants in hospital sewage water is a matter of concern, creating an endemic scenario leading to the level of an outbreak.     

Characterisation of CTX-M and AmpC genes in human isolates of Escherichia coli identified between 1995 and 2003 in England and Wales

CTX-M and AmpC genes in human isolates of Escherichia coli, their genetic environment and their host plasmids were examined. Isolates (n=103) were selected based on resistance (minimum inhibitory concentration (MIC)> or =1 microg/mL) to ceftriaxone and cefotaxime. Polymerase chain reaction (PCR) and sequencing identified 29 isolates containing bla(CTX-M-15), 1 each of bla(CTX-M-2) (a strain originating from Israel) and bla(CTX-M-40), 20 isolates containing bla(CMY-7), 4 bla(CMY-2) and 1 bla(CMY-21). This is the first study of plasmid-mediated AmpC genes in E. coli in the UK. Eleven cefoxitin-resistant, AmpC PCR-negative isolates had ampC promoter region mutations. All bla(CTX-M-15) and 24 of 25 bla(CMY) genes were associated with an ISEcp1-like element. The bla(CTX-M-2) was located in an orf513-bearing class 1 integron. Plasmid restriction digests suggest transfer of genes between different plasmid backbones.     

Occurrence of CTX-M-15- and MCR-1-producing Enterobacterales in pigs in Portugal: Evidence of direct links with antibiotic selective pressure

AimsTo undertake a prospective analysis of the occurrence of colistin-resistant and extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales colonizing pigs at two farms in Portugal, and to evaluate the putative correlations with usage of different antibiotics.Materials and methodsOne hundred and two faecal samples recovered from two different Portuguese pig farms were screened for polymyxin-resistant and ESBL-positive Enterobacterales. The authors had undertaken a study at one of the farms previously, but the use of colistin has since been banned; zinc oxide and amoxicillin are used as prophylactic and curative drugs, respectively, at this farm. The other farm included in this study used zinc oxide alone.ResultsNinety-three ESBL-producing isolates (62 Escherichia coli, 29 Klebsiella pneumoniae, one Enterobacter aerogenes and one Enterobacter cloacae) and 17 colistin-resistant isolates (12 E. coli, four K. pneumoniae and one E. cloacae) were recovered. Among the ESBL producers, the majority (84%) produced CTX-M-15, while the others produced CTX-M-1 or CTX-M-9. Many different strain and plasmid backgrounds were identified, ruling out a massive dissemination of one major clone. In total, 17 colistin-resistant isolates were recovered, all from the first farm. All produced MCR-1, corresponding to 12 E. coli (10 clones) and three K. pneumoniae (two clones). The MCR-1 producers were all recovered from the farm where colistin had been used 2 years previously.ConclusionThis study showed a surprisingly high rate of CTX-M-15 producers at two Portuguese pig farms. A link was found between antibiotic selective pressure (ß-lactam or polymyxin) and the corresponding resistance rate.     

Imported poultry meat as a source of extended-spectrum cephalosporin-resistant CMY-2-producing Salmonella Heidelberg and Salmonella Minnesota in the European Union, 2014–2015

Extended-spectrum cephalosporin (ESC)-resistant Salmonella have been described at a low level in the EU, nevertheless the increasing importation of poultry meat could be an important source of epidemic strains carrying ESC resistance genes. This study evaluated ESC resistance and its genetic platform among Salmonella isolates from poultry meat products imported into Portugal as well as clonal relatedness of the isolates. All Salmonella isolates recovered from samples of fresh meat destined for import into the EU in the scope of Portuguese official border control (2014–2015) were studied. Antibiotic susceptibility and β-lactamase production was determined by disk diffusion/microdilution. Molecular studies included detection of genes encoding acquired AmpC and extended-spectrum β-lactamases, plasmid-mediated quinolone resistance and other antibiotic resistance genes by PCR/sequencing, and clonality by MLST and XbaI-PFGE. Plasmid characterisation was assessed by conjugation assays, replicon typing (PCR-PBRT/pMLST) and hybridisation experiments (I-CeuI/S1-PFGE nuclease). Isolates belonged to Salmonella Heidelberg (n?=?6; ST15/eBG26) and Salmonella Minnesota (n?=?1; ST548/eBG77) and presented multidrug-resistant profiles, including to ESCs and/or fluoroquinolones. All but one carried bla_CMY-2, located on two epidemic plasmids, IncA/C (ST2, n?=?5) or transferable IncI1 (ST12, n?=?1). Salmonella Heidelberg was associated with five PFGE types, including one similar to an American epidemic clone. This study reveals imported poultry products as a source of uncommon and/or invasive ESC-resistant Salmonella strains in the EU. The increase of clinically relevant poultry-related serotypes in Europe must be taken into account in the current monitoring of antibiotic resistance trends and in re-evaluation of food regulations.     

Molecular characterization of novel sequence type of carbapenem-resistant New Delhi metallo-β-lactamase-1-producing Klebsiella pneumoniae in the neonatal intensive care unit of an Indian hospital

Emergence of multi-drug resistance, especially carbapenem-resistant Klebsiella pneumoniae (CRKP) is a major threat to public health. The aim of this study was to characterize CRKP isolates from infants admitted to the Neonatal Intensive Care Unit (NICU) to find the clonal outbreak of New Delhi metallo-β-lactamase (NDM) producers. In this study 17 CRKP isolates were analysed. Antimicrobial susceptibility of the isolates was determined by the disc diffusion and micro-dilution method. Carba-NP test and double-disk synergy test (DDST) were performed for the detection of carbapenemase and metallo-β-lactamase-producing K. pneumoniae. Antibiotic-resistant markers were detected by polymerase chain reaction (PCR) followed by sequencing. Clonal relatedness of the isolates was checked by multi-locus sequence typing. Conjugation experiments were performed to determine the transferability of the plasmids. All 17 CKRP isolates were found to carry bla_NDM (13 bla_NDM-1, 1 bla_NDM-4 and 3 bla_NDM-5), seven isolates carried bla_OXA-48, 13 isolates had bla_CTX-M-15, seven isolates carried bla_CMY-1 and five isolates were found to carry bla_SHV-1 on conjugative plasmids of different types (IncFIA, IncFIB, IncFIIAs, IncFIC, IncA/C, IncF, IncK, IncX, IncW and IncY). Of six different sequence types (STs) identified, ST3344 was detected as a novel ST in two K. pneumoniae isolates. Genetic environment analysis revealed ISAba125 and bleomycin genes flanking to all bla_NDM variants. This is the first report of novel ST3344 in two NDM-1-producing K. pneumoniae isolates from neonates admitted to the NICU of a North Indian Hospital. This study is provides understanding of the genetic features of this newly emerged strain type.     

Confronting Ceftolozane-Tazobactam Susceptibility in Multidrug-Resistant Enterobacterales Isolates and Whole-Genome Sequencing Results (STEP Study)

Ceftolozane-tazobactam (C/T) is frequently used for infections caused by multidrug-resistant (MDR)-Enterobacterales isolates. Whole-genome sequencing (WGS, Illumina-Hiseq 4000/NovaSeq 6000, OGC, UK) was used to study the population structure, the resistome and the virulome of C/T-susceptible and -resistant MDR Escherichia spp. (n=30) and Klebsiella spp. (n=78) isolates, recovered from lower respiratory, intra-abdominal and urinary tract infections of ICU patients from 11 Portuguese Hospitals (STEP study, 2017-2018). Minimum inhibitory concentrations (MICs) were determined (ISO-broth microdilution, breakpoints EUCAST-2020). In Escherichia spp., a weak concordance between the phenotypic and the WGS method (P=0.051) was observed in the carbapenemase detection (3/30) [bla_VIM-2 (2/3), bla_KPC-3 (1/3)]; VIM-2-Escherichia coli isolates were C/T-susceptible and only the KPC-3-Escherichia marmotae producer showed C/T-resistance. Overall, CTX-M-15-E. coli-ST131-O25:H4-H30-Rx (11/30) was the most frequent subclone, followed by CTX-M-27-E. coli-ST131-O25:H4-H30 (4/4). Moreover, a wide resistome and virulome were detected in all E. coli isolates. Among Klebsiella spp. isolates [K. pneumoniae (67/78), K. aerogenes (7/78), K. oxytoca (2/78), K. variicola (2/78)], concordance (P<0.001) was observed between the phenotypic and the genomic carbapenemase detection (21/78) [bla_KPC-3 (14/21), bla_OXA-48 (3/21), bla_OXA-181 (3/21)]. A high correlation between C/T-resistance and carbapenemase detection was established (P<0.05). Overall, a high clonal diversity was observed, mainly in KPC-3-producing K. pneumoniae isolates. An extensive resistome was detected in Klebsiella spp. isolates, whereas virulence determinants were mostly identified in carbapenemase producers (P<0.001). WGS is a powerful tool for typing characterization and microbiological study of MDR-Enterobacterales pathogens. Furthermore, carbapenemase genes are associated with C/T-resistance in Klebsiella spp., but other mechanisms might also be involved.