Carriage of genes for various extended-spectrum β-lactamases: a novel resistance strategy of Klebsiella pneumoniae in Poland

Among 110 randomly sampled strains from a collection of 247 extended-spectrum β-lactamase (ESBL)-producing clinical isolates of Klebsiella pneumoniae collected from hospitalised children in three paediatric hospitals in Poland, 64 strains (58.2%) with multiple ESBLs were found, including five non-clonal strains (4.5%) harbouring bla genes for ESBLs of three families (CTX-M, SHV and TEM). This is the first report of the emergence of triple ESBL-producing K. pneumoniae in Poland. In addition, K. pneumoniae strains harbouring bla genes for TEM-130 and TEM-132 ESBLs were detected in Poland for the first time. Epidemiological analysis of the multiple ESBL-producing K. pneumoniae isolates by pulsed-field gel electrophoresis (PFGE) revealed a relatively high genetic diversity between isolates producing the same combination of enzymes. Clonally related strains were uncommon.     

Diverse prevalence of 16S rRNA methylase genes armA and rmtB amongst clinical multidrug-resistant Escherichia coli and Klebsiella pneumoniae isolates

In this study, 112 Escherichia coli and 55 Klebsiella pneumoniae isolates with a multidrug-resistant (MDR) phenotype were collected from 2007 to 2009. All isolates simultaneously exhibited resistance to cefotaxime (or ceftazidime), ciprofloxacin (or levofloxacin) and amikacin. Plasmid-mediated 16S rRNA methylases, including armA, rmtA, rmtB, rmtC, rmtD, rmtE and npmA, were detected by polymerase chain reaction (PCR) amplification. Common β-lactamase genes, including bla_TEM, bla_SHV, bla_CTX-M, bla_PER, bla_VEB, bla_GES and bla_OXA, as well as plasmid-mediated bla_AmpC and plasmid-mediated quinolone resistance (PMQR) determinants, including qnrA, qnrB, qnrS, qepA and aac(6′)-Ib-cr, were also screened. The transferable capacity of resistance plasmids was established by conjugation testing. The genetic relatedness of isolates was analysed by pulsed-field gel electrophoresis (PFGE). Only armA and rmtB genes were detected in this study. Data showed that 93.8% of MDR E. coli and 94.5% of MDR K. pneumoniae carried at least one of armA or rmtB. The armA and rmtB genes were present in 11.6% and 82.1% of MDR E. coli, respectively. In parallel, 58.2% and 40.0% of MDR K. pneumoniae were armA- and rmtB-positive, respectively. Furthermore, the qepA gene was present in 66.3% of rmtB-carrying MDR E. coli, but it was rarely detected in MDR K. pneumoniae. Approximately 71.9% of armA-positive MDR K. pneumoniae simultaneously co-carried qnrB and bla_DHA. Moreover, 78.1% and 63.6%, respectively, of armA-positive and rmtB-positive MDR K. pneumoniae strains harboured qnr alleles and 53.1% and 59.1% harboured aac(6′)-Ib-cr. In addition, MDR E. coli strains exhibited a low prevalence of qnr alleles and aac(6′)-Ib-cr. PFGE analysis revealed divergent genetic relatedness, suggesting horizontal dissemination of armA and rmtB along with common β-lactamases and PMQR determinants amongst clinical MDR E. coli and K. pneumoniae isolates.     

In vitro activity of avibactam (NXL104) in combination with β-lactams against Gram-negative bacteria, including OXA-48 β-lactamase-producing Klebsiella pneumoniae

The objective of this study was to investigate the in vitro antibacterial activity of avibactam (formerly NXL104) in combination with imipenem, cefepime or ceftazidime against Gram-negative bacteria. Bacterial isolates included: Pseudomonas aeruginosa harbouring PER-1 β-lactamase (n = 14); Acinetobacter baumannii harbouring PER-1, OXA-51 and OXA-58 (n = 20); carbapenem-non-susceptible Klebsiella pneumoniae (n = 25) and Escherichia coli (n = 1) harbouring OXA-48; carbapenem-non-susceptible E. coli (n = 1) harbouring both IMP-1 metallo-β-lactamase and extended-spectrum β-lactamase (ESBL); carbapenem-non-susceptible Serratia marcescens (n = 1); and carbapenem-susceptible E. coli (n = 20) and K. pneumoniae isolates (n = 12) with CTX-M-15 ESBL. Minimum inhibitory concentrations (MICs) of imipenem, cefepime and ceftazidime were determined in combination with 4 mg/L avibactam by the Clinical and Laboratory Standards Institute (CLSI) method on Mueller-Hinton agar. Imipenem/avibactam and ceftazidime/avibactam displayed limited potency against A. baumannii isolates, whereas cefepime/avibactam and ceftazidime/avibactam were active against P. aeruginosa. Klebsiella pneumoniae isolates with OXA-48 β-lactamase were resistant to imipenem [MIC for 90% of the organisms (MIC₉₀) ≥4 mg/L]. MIC₉₀ values for the combination of avibactam 4 mg/L with imipenem, cefepime and ceftazidime were in the susceptible range for all strains (MIC₉₀ ≤ 0.5 mg/L). All E. coli and K. pneumoniae isolates with CTX-M-15 β-lactamase were inhibited at ≤1 mg/L for combinations with avibactam and 100% were susceptible by CLSI breakpoint criteria to imipenem, cefepime and ceftazidime. In conclusion, combinations of imipenem, cefepime and ceftazidime with avibactam may present a promising therapeutic strategy to treat infections due to K. pneumoniae with OXA-48 enzyme as well as K. pneumoniae and E. coli with CTX-M-15 enzyme.     

Activity of ceftolozane-tazobactam against Gram-negative pathogens isolated from lower respiratory tract infections in the Asia-Pacific region: SMART 2015-2016

The aim of this study was to investigate the susceptibility of respiratory Gram-negative bacteria to ceftolozane/tazobactam and other antibiotics in the Asia-Pacific region during 2015-2016. MICs were determined using the CLSI standard broth microdilution method and interpreted accordingly. Pseudomonas aeruginosa (1574 isolates), Klebsiella pneumoniae (1226), Acinetobacter baumannii (627) and Escherichia coli (476) accounted for 73.1% of 5342 Gram-negative respiratory pathogens. Susceptibility to ceftolozane/tazobactam of individual Enterobacteriaceae was >80%, except for Enterobacter cloacae (76.6%). Ceftolozane/tazobactam inhibited 81.9% of K. pneumoniae and 91.9% of E. coli, with respective MIC₅₀/MIC₉₀ values of 0.5/>32 and 0.25/2 mg/L. For carbapenem-susceptible, ESBL-producing K. pneumoniae and E. coli, susceptibility was 65.5% and 93.3%, respectively, and respective MIC₅₀/MIC₉₀ values were 2/>32 and 0.5/2 mg/L. Bla_{CTX-M-1 group} was most prevalent in selected ESBL-producing K. pneumoniae (40 of 54 isolates) and E. coli (15 of 22 isolates), with ceftolozane/tazobactam susceptibility rates of 50% and 80%, respectively. Bla_SHV-ESBL was the second most prevalent, and ceftolozane/tazobactam inhibited 20% of 20 K. pneumoniae isolates with bla_SHV-ESBL. The only effective antibiotics for carbapenem-non-susceptible K. pneumoniae (111 isolates) and E. coli (24 isolates) were amikacin and colistin. Ceftolozane/tazobactam was effective against almost all tested P. aeruginosa and carbapenem-non-susceptible strains, with susceptibility of 92.3% and 72.8%, respectively; the respective MIC₅₀/MIC₉₀ values were 1/4 and 2/>32 mg/L. The high susceptibility of ceftolozane/tazobactam remained in different age groups, patient locations, recovery times and countries, except Vietnam. In conclusion, ceftolozane/tazobactam was effective against most respiratory Gram-negative pathogens in the Asia-Pacific region; however, the emergence of carbapenem resistance mandates ongoing surveillance.     

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.     

Prediction of major antibiotic resistance in Escherichia coli and Klebsiella pneumoniae in Singapore,USA and China using a limited set of gene targets

Antibiotic resistance in Gram-negative bacteria, especially Enterobacteriaceae, can be conferred by a large number of different acquired resistance genes, although it appears that relatively few dominate. A previous survey of Escherichia coli and Klebsiella pneumoniae isolates from Sydney, Australia, revealed that a limited set of genes could reliably predict resistance to third-generation cephalosporins (3GCs) and aminoglycosides. Here we tested E. coli and K. pneumoniae isolates with a cefotaxime, ceftriaxone and/or ceftazidime minimum inhibitory concentration of ≥2 μg/mL from China and Singapore, with significantly higher resistance rates than Australia, as well as the USA. Few targets were needed to predict non-susceptibility to 3GCs (95/95; 100%) and gentamicin (47/51; 92%). The gene types detected here are consistent with previous surveys in similar countries with similar resistance rates, where the majority of 3GC resistance can be explained by bla_CTX-M genes. This study identified a limited set of genes capable of predicting resistance to 3GC and aminoglycoside antibiotics and implies a restriction in the global resistance gene pool that can be exploited for diagnostic purposes.     

Temporal evolution of polymyxin B-resistant Klebsiella pneumoniae clones recovered from blood cultures in a teaching hospital during a 7-year period

The polymyxins have become one of the last resorts to treat serious infections caused by KPC-2-producing Klebsiella pneumoniae worldwide. However, the increase of polymyxin consumption has favored the emergence of resistance to these compounds. In this study, we observed an increase in polymyxin B resistance rates from 0 to 30.6% among 224?K. pneumoniae isolates recovered from blood cultures between 2009 and 2015. Only gentamicin, tigecycline and fosfomycin remained active against the polymyxin B-resistant K. pneumoniae (PMB-R-KPN) isolates, which were classified as extensively drug-resistant (XDR; 83.3%), multidrug-resistant (MDR; 13.9%), or pan-drug resistant (2.8%). Most PMB-R-KPN clones belonged to CC258 (ST11, ST258, ST340, and ST437). A C7/ST258 XDR clone carrying distinct resistance determinants (bla_SHV-11, bla_TEM-1, bla_CTX-M-15, bla_CTX-M-14, bla_KPC-2, and rmtB-1) was introduced in 2014. Twelve of 36 PMB-R-KPN isolates showed disruption of mgrB. No mcr–1–positive isolate was found. The rapid detection of PMB-R-KPN isolates allied to implementation of effective infection control measures are of crucial importance to avoid the dissemination of high-risk PMB-R-KPN clones.     

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.     

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.     

Molecular characterisation of multiple drug-resistant Acinetobacter baumannii isolates in southern Taiwan

The purpose of this study was to develop a multiplex polymerase chain reaction (mt-PCR) assay for synchronous detection of carbapenem resistance genes and/or pandrug resistance genes in clinical isolates of multidrug-resistant Acinetobacter baumannii (MDR-AB) and to investigate the association between the genetic make-up and a drug-resistant pattern. In total, 213 MDR-AB isolates were collected. All clinical isolates underwent antimicrobial susceptibility testing and were analysed for the presence of oxacillinase genes (bla_OXA-23, bla_OXA-24, bla_OXA-51-like and bla_OXA-58), class A and C β-lactamase genes (bla_TEM-1 and bla_AmpC, respectively), and an integron-associated antibiotic resistance gene (int1) by an in-house-designed mt-PCR assay. Of the 213 isolates, 73.87% harboured both bla_TEM-1 and bla_AmpC and 83.92% carried at least three oxacillinase genes. Moreover, 64.82% of the isolates were significant in that they had two β-lactamase genes and three oxacillinase genes (P < 0.001), indicating the complexity of the genetic make-up of carbapenem-resistant A. baumannii. The bla_OXA-51-like allele was detected in the majority of these A. baumannii isolates (97.49%), whereas bla_OXA-23 was rarely prevalent in these isolates. In multivariate logistic regression, the presence of bla_OXA-23 and bla_TEM-1 had a statistically significant association with imipenem resistance [bla_OXA-23, P = 0.004, odds ratio (OR) = 10.52, 95% confidence interval (CI) 2.12–52.17; bla_TEM-1, P = 0.005, OR = 6.14, 95% CI 1.74–21.62]. These results suggest that detecting bla_OXA-23 and bla_TEM-1 genes could be used to predict imipenem resistance in MDR-AB isolates. A mt-PCR for detecting carbapenem resistance genes and pandrug resistance genes of A. baumannii isolates was developed to provide an assay to quickly screen for potential imipenem-resistant A. baumannii in the clinic.     

Spread of multidrug-resistant IncHI1 plasmids carrying ESBL gene blaCTX-M-1 and metabolism operon of prebiotic oligosaccharides in commensal Escherichia coli from healthy horses,France

The objective of the study was to identify the genetic determinants and characteristics of expanded-spectrum cephalosporin (ESC) resistance in commensal Escherichia coli from healthy horses in France in 2015. Faecal samples from 744 adult horses were screened for ESC-resistant E. coli isolates. The extended-spectrum beta-lactamase (ESBL)/AmpC resistance genes were identified using polymerase chain reaction (PCR) and sequencing. ESC phenotypes were horizontally transferred by conjugation or transformation. Plasmids carrying ESBL/AmpC genes were typed by PCR-based replicon typing, restriction fragment length polymorphism (RFLP), and plasmid multilocus sequence typing (pMLST). The ESC-resistant E. coli isolates were typed by XbaI macrorestriction analysis. Sixteen of 41 stables harboured at least one horse carrying ESC-resistant E. coli. The proportion of individually tested horses carrying ESC-resistant E. coli was 8.5% (28/328). Fifty non-redundant ESC-resistant E. coli isolates showing a great diversity of XbaI macrorestriction profiles belonged mainly to phylogroup B1, and were negative for major E. coli virulence genes, indicating they are commensal isolates. ESBL bla_CTX-M genes were dominant (bla_CTX-M-1, n=34; bla_CTX-M-2, n=8; bla_CTX-M-14, n=2) and located on conjugative plasmids belonging to various incompatibility groups (IncHI1, IncI1, IncN, IncY, or non-typeable). Among these, the multidrug-resistant IncHI1-pST9 plasmids were dominant and simultaneously harboured the bla_CTX-M-1/2 genes and an operon enabling the metabolism of short-chain fructo-oligosaccharides (scFOS). In conclusion, commensal E. coli of French horses displayed a significant distribution of IncHI1-pST9 plasmids carrying both the bla_CTX-M-1/2 gene and the fos metabolism operon. This finding highlights the risk of co-selection of multidrug-resistant IncHI1 plasmids carrying ESBL genes possibly mediated by the use of scFOS as prebiotic in horses.     

Pharmacodynamic evaluation of meropenem,cefepime, or aztreonam combined with a novel β-lactamase inhibitor,nacubactam, against carbapenem-resistant and/or carbapenemase-producing Klebsiella pneumoniae and Escherichia coli using a murine thigh-infection model

BackgroundCarbapenem-resistant Enterobacterales (CRE) and carbapenemase-producing Enterobacterales (CPE) are difficult to treat and are a serious public health threat. Nacubactam (NAC) is a novel non-β-lactam diazabicyclooctane β-lactamase inhibitor with in vitro activity against some Enterobacterales expressing classes of β-lactamases.MethodsThe antimicrobial efficacy of meropenem (MEM), cefepime (FEP), and aztreonam (ATM), each in combination with NAC, were assessed in vitro and in vivo against Klebsiella pneumoniae and Escherichia coli. Ten isolates, including CRE and/or CPE with β-lactamase genes, were used in this study. The relationship between phenotype and in vivo efficacy was assessed in a murine neutropenic thigh-infection model. Efficacy was determined by the change in bacterial quantity.ResultsThe results of the in vitro study showed the minimum inhibitory concentrations of the combination of NAC with either MEM, FEP, or ATM in a 1:1 ratio were 2 to >128-fold lower than those of MEM, FEP, or ATM alone against CRE+ isolates. In addition, combinations of β-lactams and NAC administered in the murine thigh-infection model showed greater efficacy against CRE+/CPE+, CRE+/CPE-, and CRE-/CPE+ isolates harboring various β-lactamase genes (IMP-1, IMP-6, KPC, DHA-1, or OXA-48) compared with MEM, FEP, ATM, and NAC alone.ConclusionMEM, FEP, or ATM in combination with NAC showed potent in vivo antimicrobial activity in a murine thigh-infection model caused by K. pneumoniae and E. coli, including CRE and/or CPE isolates. These findings indicate that these combinations of β-lactams and NAC are potential candidates for the treatment of CRE and/or CPE infections.     

Emergence of a ST307 clone carrying a novel insertion element MITEKpn1 in the mgrB gene among carbapenem-resistant Klebsiella pneumoniae from Moscow,Russia

Carbapenem-resistant Klebsiella pneumoniae (CRKP) represents a major nosocomial pathogen with only a few antimicrobial agents, including colistin, remaining active. However, the emergence of colistin-resistant (Col-R) isolates is compromising the activity of colistin. In this study, a collection of 159 CRKP recovered from three hospitals in Moscow (Russian Federation) was examined. The isolates demonstrated resistance to cephalosporins (100%), ciprofloxacin (92.5%), fosfomycin (90.1%), netilmicin (81.1%), gentamicin (84.3%) and amikacin (49.7%). The rate of colistin resistance (MIC > 2 mg/L by broth microdilution) was 44.7%; moreover, 6.7% of isolates were tigecycline-resistant. Among 18 sequence types (STs) discovered, isolates of five lineages including ST307 (n = 46; 28.9%), ST395 (n = 40; 25.2%), ST377 (n = 17; 10.7%), ST48 (n = 17; 10.7%) and ST23 (n = 16; 10.1%) dominated. Carriage of a bla_OXA-48-like carbapenemase gene was detected in 146 CRKP (91.8%); 11 (6.9%) and 2 (1.3%) isolates harboured bla_NDM-1 and bla_KPC-3, respectively. Among 71 Col-R isolates, colistin MICs ranged from 4 mg/L to >1024 mg/L (MIC_50/90, 2/512 mg/L). All Col-R isolates were mcr-1-negative. In 19 (26.8%) Col-R isolates, inactivation of mgrB by insertion sequences IS1A, IS1R, ISKpn14 and ISKpn26 and a novel miniature inverted-repeat transposable element (MITE) Kpn1 was observed. Carriage of MITEKpn1 was restricted to six ST307 isolates and affected mgrB at nucleotide position 75. mgrB deletion was observed in four (5.6%) Col-R isolates. Moreover, PmrA and/or PmrB were altered in three (4.5%) Col-R isolates with wild-type mgrB. Thus, bla_OXA-48-like-carrying Col-R ST307 K. pneumoniae is emerging as a dominant clone in Moscow.     

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.     

In Vitro Efficacy of Meropenem-Cefmetazole Combination Therapy against New Delhi Metallo-β-lactamase-producing Enterobacteriaceae

BackgroundLimited treatment options complicate management of infections with New Delhi metallo-β-lactamase (NDM)-producing organisms. The efficacy of combination therapy with meropenem (MEM) and cefmetazole (CMZ) was assessed against NDM-producing Enterobacteriaceae.Materials and MethodsTwelve Escherichia coli clinical isolates harbouring bla_NDM-1 and a positive control E. coli BAA-2469 harbouring bla_NDM-1 were studied. Minimum inhibitory concentrations (MICs) of MEM, ertapenem (ERT) and CMZ were determined by broth microdilution. Checkerboard and time-kill assays were performed to confirm the in vitro efficacy of the MEM/CMZ combination. Scanning electron microscopy, kinetic studies and whole-genome sequence analysis were used to determine the antimicrobial resistance mechanisms.ResultsMICs of MEM, ERT and CMZ in monotherapy ranged from 8 to 32, 16 to 128, and 32 to 512 µg/mL, respectively. In the checkerboard assay, MEM/ERT resulted in no synergy, whereas MEM/CMZ showed a synergistic effect in all the tested isolates. Furthermore, the MIC of MEM in combination decreased by 2- to 8-fold compared with that of MEM alone. The time-kill study revealed a bactericidal effect in 4 of 13 isolates at 24 h. Scanning electron microscopy showed spheroidisation of the bacterial cell in the MEM/CMZ combination; this was not observed in single antibiotic conditions. Kinetic studies indicated CMZ was a better antagonist for NDM-1 than ERT. Whole-genome sequence analysis did not reveal any explainable differences between isolates susceptible and those non-susceptible to combination therapy.ConclusionIn vitro studies showed the potential effectiveness of MEM/CMZ combination therapy against NDM-producing organisms.     

Activity of ceftazidime-avibactam alone and in combination with polymyxin B against carbapenem-resistant Klebsiella pneumoniae in a tandem in vitro time-kill/in vivo Galleria mellonella survival model analysis

Ceftazidime-avibactam is used clinically in combination with a polymyxin for the treatment of carbapenem-resistant Gram-negative infections; however, there are limited data to support this practice. The objective of this study was to evaluate the activity of ceftazidime-avibactam and polymyxin B alone and in combination against Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae in a tandem in vitro time-kill/in vivo Galleria mellonella survival model assay. Three KPC-3-producing K. pneumoniae clinical isolates were used for all experiments. All isolates harbored mutations in ompk35 and one isolate in ompk36; two isolates were susceptible to both ceftazidime-avibactam and polymyxin B, and one was resistant to both. Ceftazidime-avibactam was bactericidal against 2 of 3 strains at ≥2x minimum inhibitory concentration (MIC) whereas polymyxin B was not bactericidal against any strain at any concentration. Combinations at 1/4x or 1/2x MIC were not bactericidal or synergistic against any of the 3 isolates. In survival experiments, ceftazidime-avibactam at 4x MIC significantly improved larval survival over the untreated control strain whereas polymyxin B at 4x MIC did not. Combining polymyxin B with ceftazidime-avibactam at 4x MIC did not improve survival compared to ceftazidime-avibactam alone. This work indicates there is no improvement in in vitro bactericidal activity or in vivo efficacy when polymyxin B is combined with ceftazidime-avibactam against KPC-producing K. pneumoniae. This combination should be avoided in lieu of ceftazidime-avibactam alone or other potentially more efficacious, less toxic combination regimens.     

In vitro activity of the siderophore monosulfactam BAL30072 against contemporary Gram-negative pathogens from New York City,including multidrug-resistant isolates

The in vitro activity of BAL30072 was assessed against clinical isolates from NYC hospitals, including isolates from a citywide surveillance study and a collection of isolates with well-characterised resistance mechanisms. BAL30072 was the most active β-lactam against Pseudomonas aeruginosa (MIC_50/90, 0.25/1 μg/mL), Acinetobacter baumannii (MIC_50/90, 4/>64 μg/mL) and KPC-possessing Klebsiella pneumoniae (MIC_50/90, 4/>64 μg/mL). Combining BAL30072 with meropenem resulted in a ≥4-fold decrease in the BAL30072 MIC₉₀ both for A. baumannii and K. pneumoniae. For isolates with a BAL30072 MIC > 4 μg/mL, addition of a sub-MIC concentration of colistin resulted in a four-fold decrease in the BAL30072 MIC in 44% of P. aeruginosa, 82% of A. baumannii and 23% of K. pneumoniae. Using sub-MIC concentrations, BAL30072 plus colistin was bactericidal against 4 of 11 isolates in time–kill studies. BAL30072 MICs were frequently lower for P. aeruginosa and K. pneumoniae when tested using Mueller–Hinton agar versus Iso-Sensitest agar or Mueller–Hinton broth. Against the well-characterised isolates, reduced susceptibility to BAL30072 correlated with mexA and mexX expression (P. aeruginosa), adeB expression (A. baumannii) and presence of SHV-type ESBLs (A. baumannii and K. pneumoniae). BAL30072 shows promising activity against contemporary Gram-negatives, including MDR P. aeruginosa, A. baumannii and K. pneumoniae. Enhanced activity was often present when BAL30072 was combined with meropenem or colistin. BAL30072 MICs were influenced by the testing method, particularly for P. aeruginosa and K. pneumoniae. Further in vivo studies are warranted to determine the potential clinical utility of BAL30072 alone and combined with other agents.     

A novel family (QnrAS) of plasmid-mediated quinolone resistance determinant

The spread of plasmid-mediated quinolone resistance determinants (qnr-like determinants) was evaluated in a collection of 232 ciprofloxacin-resistant or extended-spectrum β-lactamase (ESBL)-producing enterobacterial isolates recovered between November 2007 and May 2008 at Padua University Hospital, Italy. qnr genes were mainly found in Klebsiella pneumoniae (68%) and to a lesser extent in Escherichia coli (5.1%). Among the qnrA1, qnrS1 and qnrB19 alleles found, the latter was by far the most frequent. Genetic environment analysis revealed that one qnrB19 gene in E. coli was embedded in an ISCR1 complex class 1 integron. All other qnrB19 genes were flanked by an ISEcp1C region as part of the Tn2012 transposon. qnrA1- and qnrS1-containing strains were not clonally related. Both topoisomerase II mutations and ESBL (mainly SHV-12, TEM-1 and TEM-150 types) were present in most of the qnr-positive strains. qnrB19 is extremely frequent in K. pneumoniae isolates from Italy. In addition, association of qnrB19 with the ISCR1 mobile element in E. coli suggests a broad distribution of this resistance gene in the near future.     

KPC-2-producing Klebsiella pneumoniae ST147 in a neonatal unit: Clonal isolates with differences in colistin susceptibility attributed to AcrAB-TolC pump

This study characterizes four KPC-2-producing Klebsiella pneumoniae isolates from neonates belonging to a single sequence type 147 (ST147) in relation to carbapenem resistance and explores probable mechanisms of differential colistin resistance among the clonal cluster.Whole genome sequencing (WGS) revealed that the isolates were nearly 100% identical and harbored resistance genes (bla_KPC-2,_OXA-9,_CTX-M-15,_SHV-11,_OXA-1,_TEM-1B, oqxA, oqxB, qnrB1, fosA, arr-2, sul1, aacA4, aac(6′)Ib-cr, aac(6′)Ib), and several virulence genes. bla_KPC-2 was the only carbapenem-resistant gene found, bracketed between ISKpn7 and ISKpn6 of Tn4401b on a non-conjugative IncFII plasmid. Remarkably, one of the clonal isolates was resistant to colistin, the mechanistic basis of which was not apparent from comparative genomics. The transmissible colistin resistance gene, mcr, was absent. Efflux pump inhibitor, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) rendered a 32-fold decrease in the minimum inhibitory concentration (MIC) of colistin in the resistant isolate only. acrB, tolC, ramA, and soxS genes of the AcrAB-TolC pump system overexpressed exclusively in the colistin-resistant isolate, although the corresponding homologs of the AcrAB-TolC pump, regulators and promoters were mutually identical. No change was observed in the expression of other efflux genes (kpnE/F and kpnG/H) or two-component system (TCS) genes (phoP/phoQ, pmrA/pmrB).Colistin resistance in one of the clonal KPC-2-producing isolates is postulated to be due to overexpression of the AcrAB-TolC pump. This study is probably the first to report clinical clonal K. pneumoniae isolates with differences in colistin susceptibility. The presence of carbapenem-resistant isolates with differential behavior in the expression of a genomically identical pump system indicates the nuances of the resistance mechanisms and the difficulty of treatment thereof.