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Rollout of meningococcal serogroup A conjugate vaccine in Africa started in 2010, aiming to eliminate meningitis outbreaks, in meningitis belt countries. Since then, studies have been conducted, primarily using isolates, to assess the vaccine impact on the distribution of meningococcal strains in the region. Here, we implemented an innovative, culture-free whole-genome sequencing approach on almost 400 clinical specimens collected between 2017 and 2019 from meningococcal meningitis cases in 6 African countries. About 50% of specimens provided high-quality whole-genome sequence data for comprehensive molecular profiling of the meningococcal pathogen. Three major clonal complexes were identified: CC11 associated with serogroup W, CC181 associated with serogroup X, and CC10217 associated with serogroup C, which continues to rise as a predominant clonal complex in the region. Genomic surveillance for meningococcal meningitis can be significantly improved using culture-free methods to increase data representativeness and monitor changes in epidemiological landscape, especially for countries with low culture rate.

Bacillus anthracis, the causative agent of anthrax, is a high-consequence bacterial pathogen that occurs naturally in many parts of the world and is considered an agent of biowarfare or bioterrorism. Understanding antimicrobial susceptibility profiles of B. anthracis isolates is foundational to treating naturally occurring outbreaks and to public health preparedness in the event of an intentional release. In this systematic review, we searched the peer-reviewed literature for all publications detailing antimicrobial susceptibility testing of B. anthracis. Within the set of discovered articles, we collated a subset of publications detailing susceptibility testing that followed standardized protocols for Food and Drug Administration–approved, commercially available antimicrobials. We analyzed the findings from the discovered articles, including the reported minimal inhibitory concentrations. Across the literature, most B. anthracis isolates were reported as susceptible to current first-line antimicrobials recommended for postexposure prophylaxis and treatment. The data presented for potential alternative antimicrobials will be of use if significant resistance to first-line antimicrobials arises, the strain is bioengineered, or first-line antimicrobials are not tolerated or available.

Current antimicrobial treatment recommendations for melioidosis, the disease caused by Burkholderia pseudomallei, are largely based on studies of strains isolated from the Eastern Hemisphere (EH), ...

Human anthrax cases necessitate rapid response. We completed Bacillus anthracis nanopore whole-genome sequencing in our high-containment laboratory from a human anthrax isolate hours after receipt. The de novo assembled genome showed no evidence of known antimicrobial resistance genes or introduced plasmid(s). Same-day genomic characterization enhances public health emergency response.

Current antimicrobial treatment recommendations for melioidosis, the disease caused by

Infections in immunocompromised patients that are caused by extensively drug-resistant (XDR) Acinetobacter baumannii strains have been increasingly reported worldwide. In particular, carbapenem-resistant A. baumannii strains are a prominent cause of health care-associated infections. Here, we report draft genome assemblies for two clinical XDR A. baumannii isolates obtained from hospitalized patients in Pakistan.

Shigella spp. are the most common cause of dysentery in developing countries and the second leading cause of diarrheal deaths worldwide. Multidrug-resistant (MDR) Shigella spp. are a serious threat to global health. Herein, we report draft genome sequences for three MDR Shigella isolates from Pakistan, two Shigella flexneri isolates and one Shigella sonnei isolate.

Shigella spp. are the most common cause of dysentery in developing countries and the second leading cause of diarrheal deaths worldwide. Multidrug-resistant (MDR) Shigella spp. are a serious threat to global health. Herein, we report draft genome sequences for three MDR Shigella isolates from Pakistan, two Shigella flexneri isolates and one Shigella sonnei isolate.

Bacillus anthracis, the etiologic agent of anthrax, is characteristically susceptible to penicillin despite containing two chromosomal β-lactamase genes. Few naturally occurring penicillin-resistant B. anthracis isolates have been reported., Bacillus anthracis, the etiologic agent of anthrax, is characteristically susceptible to penicillin despite containing two chromosomal β-lactamase genes. Few naturally occurring penicillin-resistant B. anthracis isolates have been reported. Here, we report the draft genome sequences for three penicillin-resistant B. anthracis strains, strain 32, UT308, and SK57.

Penicillin (PEN) is a low-cost option for anthrax treatment, but naturally occurring resistance has been reported. β-Lactamase expression (bla1, bla2) in Bacillus anthracis is regulated by a sigma factor (SigP) and its cognate anti-sigma factor (RsiP). Mutations leading to truncation of RsiP were previously described as a basis for PEN resistance. Here, we analyze whole-genome sequencing (WGS) data and compare the chromosomal sigP-bla1 regions from 374 B. anthracis strains to determine the frequency of mutations, identify mutations associated with PEN resistance, and evaluate the usefulness of WGS for predicting PEN resistance. Few (3.5%) strains contained at least 1 of 11 different mutations in sigP, rsiP, or bla1. Nine of these mutations have not been previously associated with PEN resistance. Four strains showed PEN resistance (PEN-R) by conventional broth microdilution, including 1 strain with a novel frameshift in rsiP. One strain that carries the same rsiP frameshift mutation as that found previously in a PEN-R strain showed a PEN-susceptible (PEN-S) phenotype and exhibited decreased bla1 and bla2 transcription. An unexpectedly small colony size, a reduced growth rate, and undetectable β-lactamase activity levels (culture supernatant and cell lysate) were observed in this PEN-S strain. Sequence analysis revealed mutations in genes associated with growth defects that may contribute to this phenotype. While B. anthracisrsiP mutations cannot be exclusively used to predict resistance, four of the five strains with rsiP mutations were PEN-R. Therefore, the B. anthracissigP-bla1 region is a useful locus for WGS-based PEN resistance prediction, but phenotypic testing remains essential. IMPORTANCE Determination of antimicrobial susceptibility of B. anthracis is essential for the appropriate distribution of antimicrobial agents for postexposure prophylaxis (PEP) and treatment of anthrax. Analysis of WGS data allows for the rapid detection of mutations in antimicrobial resistance (AMR) genes in an isolate, but the presence of a mutation in an AMR gene does not always accurately predict resistance. As mutations in the anti-sigma factor RsiP have been previously associated with high-level penicillin resistance in a limited number of strains, we investigated WGS assemblies from 374 strains to determine the frequency of mutations and performed functional antimicrobial susceptibility testing. Of the five strains that contained mutations in rsiP, only four were PEN-R by functional antimicrobial susceptibility testing. We conclude that while sequence analysis of this region is useful for AMR prediction in B. anthracis, genetic analysis should not be used exclusively and phenotypic susceptibility testing remains essential.

Clinical outcomes of melioidosis patients improve when the infecting agent, Burkholderia pseudomallei , is rapidly detected and identified by laboratory testing. Detection of B. pseudomallei DNA or recovery of the pathogen by culture from urine can support a diagnosis of melioidosis and guide patient care. Two new methods, designated filter-capture DNA isolation (FCDI) and filter cellular recovery (FCR), were developed to increase the sensitivity of detection and recovery of viable B. pseudomallei cells from small volumes (0.45 ml) of urine. DNA from eight strains of B. pseudomallei that were spiked into synthetic urine at low concentrations (1 × 10 2 CFU/ml) was detected in FCDI cell lysates using real-time PCR with greater consistency than with preparations from a QIAamp DNA Blood minikit. The FCR method showed greater B. pseudomallei detection sensitivity than conventional urine culture methods and resulted in typical colony growth at 24 h from as few as 1 × 10 2 CFU/ml. In addition, the FCR method does not rely on precipitation of a urine pellet by centrifugation and requires a smaller volume of urine. The FCDI and FCR methods described here could improve time-to-results and decrease the number of negative B. pseudomallei reports that are currently observed from urine culture as a consequence of samples containing low or variable bacterial cell concentrations.

Streptococcus pneumoniae remains a leading cause of disease in children and adults. Serotypes differ in invasiveness, virulence, and antibiotic resistance; therefore, serotype surveillance is necessary to monitor the burden of pneumococcal disease, especially in the setting of pneumococcal vaccination programs. The Tigecycline Evaluation Surveillance Trial, (TEST), is an on-going global antibiotic susceptibility surveillance program. Serotypes and antibiotic susceptibilities of 2173 invasive S. pneumoniae in this existing database during 2004-2008 were evaluated. Worldwide, serotypes 19A (28%), 19F (10%) and 14 (9%) were the most common in children under 5 years. In adults over 16 years, 19A (13%), 3, 6A and 7F (all 7%) were most common. Serotypes 19A, 6A, 19F, 6B, 15A, 9V, and 14 exhibited significantly higher levels of erythromycin resistance (P0.05), while 19A, 19F, 35B, 6A, 6B, 23A, 9V, 15A, and 14 demonstrated higher rates of penicillin resistance (P0.05). This analysis of an existing pathogen database provides a snapshot of global serotype data and describes the consequential issue of antibiotic resistance in specific serotypes, many of which are increasingly common causes of invasive pneumococcal disease.

The emergence and spread of colistin resistance among multidrug-resistant (MDR) Klebsiella pneumoniae represent a critical threat to global health. Here, we report the complete genome sequences of 10 MDR, colistin-susceptible and -resistant K. pneumoniae clinical isolates obtained in Pakistan between 2010 and 2013.

In 2009-2010, 3646 urinary tract isolates of Enterobacteriaceae spp. were isolated from hospitalized patients in North America and Europe. Extended-spectrum beta-lactamase (ESBL) production was detected in 8.5% and 8.8% of Escherichia coli and Klebsiella pneumoniae, respectively, in North America and in 17.6% and 38.9% for Europe, respectively. The carbapenems (ertapenem and imipenem) were the most active agents in vitro, with ampicillin-sulbactam the least active. Molecular characterization of about 50% of ESBL-positive isolates identified the presence of bla(CTX-M) genes in over 90% of Escherichia coli from both continents. bla(KPC) was more common in North American isolates of K. pneumoniae than in European isolates (21.4% versus 6.9%). bla(TEM) and AmpC genes were infrequent. Enterobacteriaceae spp. isolated from hospitalized patients with urinary tract infections in both North America and Europe are often resistant to commonly used antimicrobials with bla(CTX-M) genes common in both Escherichia coli and K. pneumoniae.

The worldwide dissemination of extended-spectrum-β-lactamase (ESBL)- and carbapenemase-producing Enterobacteriaceae is a major concern in both hospital and community settings. Rapid identification of these resistant pathogens and the genetic determinants they possess is needed to assist in clinical practice and epidemiological studies. A collection of Escherichia coli , Klebsiella pneumoniae , Klebsiella oxytoca , and Proteus mirabilis isolates, including phenotypically ESBL-positive ( n = 1,093) and ESBL-negative isolates ( n = 59), obtained in 2008–2009 from a longitudinal surveillance study (SMART) was examined using an in vitro nucleic acid-based microarray. This approach was used to detect and identify bla ESBL ( bla SHV , bla TEM , and bla CTX-M genes of groups 1, 2, 9, and 8/25) and bla KPC genes and was combined with selective PCR amplification and DNA sequencing for complete characterization of the bla ESBL and bla KPC genes. Of the 1,093 phenotypically ESBL-positive isolates, 1,041 were identified as possessing at least one bla ESBL gene (95.2% concordance), and 59 phenotypically ESBL-negative isolates, used as negative controls, were negative. Several ESBL variants of bla TEM ( n = 5), bla SHV ( n = 11), bla CTX-M ( n = 19), and bla KPC ( n = 3) were detected. A new bla SHV variant, bla SHV-129 , and a new bla KPC variant, bla KPC-11 , were also identified. The most common bla genes found in this study were bla CTX-M-15 , bla CTX-M-14 , and bla SHV-12 . Using nucleic acid microarrays, we obtained a “molecular snapshot” of bla ESBL genes in a current global population; we report that CTX-M-15 is still the dominant ESBL and provide the first report of the new β-lactamase variants bla SHV-129 and bla KPC-11 .

The emergence and dissemination of carbapenemases, bacterial enzymes able to inactivate most β-lactam antibiotics, in Enterobacteriaceae is of increasing concern. The concurrent spread of resistance against colistin, an antibiotic of last resort, further compounds this challenge further. Whole-genome sequencing (WGS) can play a significant role in the rapid and accurate detection/characterization of existing and emergent resistance determinants, an essential aspect of public health surveillance and response activities to combat the spread of antimicrobial resistant bacteria. In the current study, WGS data was used to characterize the genomic content of antimicrobial resistance genes, including those encoding carbapenemases, in 10 multidrug-resistant Klebsiella pneumoniae isolates from Pakistan. These clinical isolates represented five sequence types: ST11 (n = 3 isolates), ST14 (n = 3), ST15 (n = 1), ST101 (n = 2), and ST307 (n = 1). Resistance profiles against 25 clinically-relevant antimicrobials were determined by broth microdilution; resistant phenotypes were observed for at least 15 of the 25 antibiotics tested in all isolates except one. Specifically, 8/10 isolates were carbapenem-resistant and 7/10 isolates were colistin-resistant. The blaNDM-1 and blaOXA-48 carbapenemase genes were present in 7/10 and 5/10 isolates, respectively; including 2 isolates carrying both genes. No plasmid-mediated determinants for colistin resistance (e.g. mcr) were detected, but disruptions and mutations in chromosomal loci (i.e. mgrB and pmrB) previously reported to confer colistin resistance were observed. A blaOXA-48-carrying IncL/M-type plasmid was found in all blaOXA-48-positive isolates. The application of WGS to molecular epidemiology and surveillance studies, as exemplified here, will provide both a more complete understanding of the global distribution of MDR isolates and a robust surveillance tool useful for detecting emerging threats to public health.

Rapid methods to determine antimicrobial susceptibility would assist in the timely distribution of effective treatment or postexposure prophylaxis in the aftermath of the release of bacterial biothreat agents such as Bacillus anthracis , Yersinia pestis , or Burkholderia pseudomallei . Conventional susceptibility tests require 16 to 48 h of incubation, depending on the bacterial species. We evaluated a method that is based on laser light scattering technology that measures cell density in real time. We determined that it has the ability to rapidly differentiate between growth (resistant) and no growth (susceptible) of several bacterial threat agents in the presence of clinically relevant antimicrobials. Results were available in B. anthracis and Y. pestis and B. pseudomallei . One exception was B. pseudomallei in the presence of ceftazidime, which required >10 h of incubation. Use of laser scattering technology decreased the time required to determine antimicrobial susceptibility by 50% to 75% for B. anthracis , Y. pestis , and B. pseudomallei compared to conventional methods.

Rapid identification of patients infected with clarithromycin-resistant Helicobacter pylori without the need for culture can help to avoid useless prescriptions of clarithromycin. We developed and tested a routine real-time quantitative PCR assay dedicated to that purpose. One hundred ninety-six consecutive gastric biopsy specimens were examined by culture, histology performed by a trained physician, and rapid PCR with the LightCycler apparatus. Infection was defined as (i) positivity of culture, (ii) positivity of histology, or (iii) positivity of PCR if confirmed by positivity of a concomitant indirect test (serology or urea breath test). Susceptibility to clarithromycin was tested by E-test and PCR. The prevalence of infection was 33.7% (66 of 196 samples). The sensitivities of culture, histology, and PCR were 90.9% (60 of 66 samples), 87.9% (58 of 66 samples), and 97.0% (64 of 66 samples), respectively. The specificity of PCR was 94.6% (123 of 130 samples). The linearity of the PCR results was achieved over a 6-log range of input DNA, and we were able to accurately quantify as few as 300 bacteria and to qualitatively detect as few as 30 bacteria per DNA sample. For clarithromycin susceptibility testing, there was 98.2% (55 of 56 samples) concordance between E-test and PCR. Forty-eight strains were clarithromycin susceptible, and 9 strains were clarithromycin resistant. The single discrepancy concerned a culture which was a mixture of mutant and wild type, with a susceptible-to-resistant ratio of 11.5: the resistant population was detected by E-test but not by PCR. Our PCR assay is accurate for fast detection of H. pylori as well as of clarithromycin resistance and is also able to objectively determine bacterial density.

The Study for Monitoring Antimicrobial Resistance Trends has been monitoring the activity of antimicrobials indicated for the treatment of intra-abdominal infections since 2004. This report documents the in vitro activity of several recommended antimicrobials against 3449 gram-negative bacilli isolated from the 30 and 25 participating sites in North America in 2010–2011, respectively, and characterizes the extended-spectrum beta-lactamases (ESBL) identified in ESBL-positive and ertapenem-non-susceptible isolates of Enterobacteriaceae. Escherichia coli , Klebsiella pneumoniae , Enterobacter cloacae , Proteus mirabilis , Klebsiella oxytoca , Citrobacter freundii , Enterobacter aerogenes , Serratia marcescens , and Morganella morgannii were the most common species isolated. The incidence of beta-lactamase production was 8.8% and 8.9% for E. coli and K. pneumoniae , respectively. Overall the most active antimicrobials were amikacin, piperacillin-tazobactam, imipenem, and ertapenem, although beta-lactamase production reduced the activity of most agents. Characterization of beta-lactamase genes determined that bla SHV , bla CTX-M , bla AmpC , and bla KPC were commonly found in most beta-lactamase–positive isolates.

A study was designed to characterize 35 non-repeat isolates of VIM- and IMP-producing Enterobacteriaceae obtained from the SMART surveillance program. Characterization was done by polymerase chain reaction, sequencing, and multi-locus sequencing. The VIM-1, -2, -5, -26, -27, -33, and IMP-1 and -26–producing Enterobacteriaceae were obtained from Greece, Italy, Spain, Philippines, Turkey, Australia, Mexico, USA, and India. Plasmids varied in size from 60 to 300 kb and belonged to IncA/C, IncF, IncHI1, IncL/M, IncN, and IncK incompatibility groups. The most common gene cassettes consisted of bla IMP-26 , qacG , aacA4 and bla VIM , aacA7 , dhfrI , and aadA1 . Intercountry, interhospital, intrahospital, interspecies, and intraclonal spread of bla VIM and bla IMP containing plasmids and sequence types (STs) occurred in Greece, Italy, Spain, and Philippines. ST147 with IncA/C and IncF plasmids is an important drug-resistant ST among Klebsiella pneumoniae with VIMs. Our study highlights the importance of surveillance programs using molecular techniques as powerful tools to identify the transmission of STs with their respective plasmids.

A study was designed to characterize nonrepeat isolates of carbapenemase-producing K. pneumoniae obtained from the SMART worldwide surveillance program during 2008 and 2009. Characterization was done by PCR and sequencing for bla VIM , bla IMP , bla NDM , bla OXA , bla KPC , and plasmid-mediated quinolone resistance and virulence factors (VFs). Genetic relatedness was determined with pulsed-field gel electrophoresis (PFGE) using XbaI and multilocus sequence typing. A total of 110 isolates were included; 47 possess genes that encode K. pneumoniae carbapenemases (KPCs), 26 NDMs, 19 VIMs, 13 OXA-48-like, and 5 imipenems (IMPs). We identified 3 different major sequence types (STs) among 65% of the isolates (i.e., ST11 [ n = 11], ST147 [ n = 23], and ST258 [ n = 38]). ST11 and ST147, producing OXA-48-like and NDMs, were found in Argentina, Turkey, Greece, Italy, and India; ST258, producing KPCs, was present in the United States, Israel, Greece, and Puerto Rico. The major STs consisted of up to 4 different pulsotypes, and each pulsotype had a specific geographical distribution. A new ST, named ST903, with bla IMP-26 , was identified in the Philippines, while two bla OXA-48 -positive isolates were detected in the United States. There were no significant differences in the distribution of the VFs between the isolates; all were positive for fimH , mrkD , wabG , and ureA . This is the first report of OXA-48-like enzymes in North America. Our study highlights the importance of surveillance programs using molecular techniques as powerful tools to identify the importance of international sequence types.

A study was designed to evaluate the modified Hodge test (MHT), Mastdiscs ID inhibitor combination disks (MDI), Rosco Diagnostica Neo-Sensitabs (RDS), metallo-β-lactamase (MBL) Etest, and in-house multiplex PCR for the detection of well-characterized carbapenemase-producing Enterobacteriaceae . One hundred forty-two nonrepeat clinical isolates of carbapenemase-producing Enterobacteriaceae (including Klebsiella spp., Escherichia coli , Citrobacter freundii , and Enterobacter spp.) obtained from the SMART worldwide surveillance program during 2008 to 2009 were included. These included 49 KPC-, 27 NDM-, 19 VIM-, 14 OXA-48-like enzyme-, and 5 IMP-producing isolates and 28 carbapenem-resistant, carbapenemase-negative isolates. The manufacturer's instructions were followed for MDI, RDS, and MBL Etest and CLSI guidelines for MHT. A multiplex PCR was designed to detect KPC, NDM, VIM, IMP, and OXA-48-like carbapenemases. Overall, the sensitivity and specificity were 78% and 93% for MDI, 80% and 93% for RDS, 58% and 93% for MHT, and 55% and 100% for MBL Etest, respectively. The PCR had 100% sensitivity and specificity. MDI and RDS performed well for the detection of KPCs and NDMs but poorly for VIMs, IMPs, and OXA-48-like enzymes. MHT performed well for KPCs and OXA-48-like enzymes but poorly for NDMs, VIMs, and IMPs. MDI and RDS were easy to perform and interpret but lacked sensitivity for OXA-48-like enzymes, VIMs, and IMPs. MHT and MBL Etest were often difficult to interpret. We recommend using molecular tests for the optimal detection of carbapenemase-producing Enterobacteriaceae .

The Study for Monitoring Antimicrobial Resistance Trends is an ongoing multi-year surveillance study that tracks worldwide antimicrobial resistance trends among aerobic and facultatively anaerobic Gram-negative bacilli isolated from intra-abdominal infections. During 2008-2009, 1366 isolates of Escherichia coli were collected from 19 investigator sites in 11 Latin American countries. Of the 1366 isolates, 323 (23.6%) were extended spectrum beta-lactamase (ESBL)-positive. Overall, the most effective agents tested were imipenem, ertapenem, and amikacin with susceptibilities of ≥96%. Against ESBL-positive isolates, only imipenem and ertapenem exhibited susceptibility ≥90%. Based on the use of the new Clinical and Laboratory Standards Institute clinical breakpoints for ertapenem (resistance ≥1 μg/ml), resistance to ertapenem among all E. coli isolates was only 0.3% (4/1366) throughout the region, ranging from 0% in several countries up to 1.2% in Ecuador. Against ESBL-positive isolates only, resistance to ertapenem in Latin America overall was 0.9% (3/323), with a maximum of 9.1% (1/11) observed in Argentina.

A total of 2,841 clinical isolates of Klebsiella pneumoniae from intra-abdominal infections worldwide were collected in the Study for Monitoring Antimicrobial Resistance Trends (SMART) during 2008 and 2009. Overall, 22.4% of isolates had extended-spectrum β-lactamases (ESBLs). The most active antibiotics among the 11 tested were imipenem, amikacin, and ertapenem, though even these, like all other comparators, were less consistently active against ESBL-positive isolates than against ESBL-negative isolates. Globally, 6.5% of isolates were ertapenem resistant based on the June 2010 clinical breakpoints published by the Clinical and Laboratory Standards Institute, with MICs of ≥1 μg/ml. Molecular characterization of 43 isolates with ertapenem MICs of ≥4 μg/ml showed that they variously produced CTX-M or SHV ESBLs combined with altered impermeability and/or had KPC ( n = 28), OXA-48 ( n = 3), or VIM ( n = 1) carbapenemases. Further monitoring of ertapenem susceptibility and molecular characterization of ertapenem-resistant isolates are needed.

To investigate the β-lactamase background of ertapenem non-susceptible isolates for the presence of the most commonly detected carbapenemase genes, bla(KPC), bla(OXA-48) and bla(VIM), and the newly described bla(NDM-1).Two hundred and thirty-five ertapenem-non-susceptible (MIC ≥ 0.5 mg/L) isolates of Enterobacteriaceae from the worldwide Study for Monitoring Antimicrobial Resistance Trends (SMART) 2009 programme were screened using a multiplex PCR for the presence of bla(KPC), bla(OXA-48), bla(VIM) and bla(NDM-1) genes. Extended-spectrum β-lactamase (ESBL) and AmpC genes (bla(ESBL) and bla(AmpC)) were identified using the Check-MDR CT101 microarray. DNA sequencing was performed to identify the bla(ESBL), bla(KPC) and bla(NDM-1) genes. Molecular typing was also performed to genetically characterize these isolates.Sixty-six isolates (28%) had a carbapenemase gene, with bla(NDM-1) identified in 33 isolates including 2 isolates carrying both bla(NDM-1) and bla(OXA-48); other carbapenemase genes found included bla(KPC) (n = 23), bla(VIM) (n = 7) and bla(OXA-48) (n = 3). All bla(NDM-1)-carrying isolates were from patients in India and comprised five different species. With the exception of one isolate carrying only bla(NDM-1), all bla(NDM-1) carbapenemase-possessing isolates carried additional β-lactamases in various combinations: bla(ESBL) and bla(AmpC) (n = 18); bla(ESBL) (n = 10); bla(ESBL), bla(AmpC) and bla(OXA-48) (n = 2); and bla(AmpC) (n = 2). Except for bla(OXA-48)-carrying isolates, novel multilocus sequence types or enterobacterial repetitive intergenic consensus PCR patterns were observed along with clonal dissemination within and among sites.A range of carbapenemase genes, associated with diverse ESBLs and/or AmpC backgrounds, were found among Enterobacteriaceae isolated during the study. Many of these ertapenem non-susceptible strains were clonally related and carried various combinations of β-lactamases.

A total of 3160 clinical isolates of Escherichia coli from intra-abdominal infections were collected during 2008–2009 from 13 European countries. The frequency of extended-spectrum β-lactamase (ESBL)-producing isolates in Europe was 11%. The most active antibiotics tested were typically imipenem, ertapenem, and amikacin, although the activity of all non-carbapenem antibiotics was lower when tested against ESBL-positive isolates than when tested against ESBL-negative isolates. Ertapenem exhibited 99.3% susceptibility with all isolates, and 96.8% susceptibility with ESBL-positive isolates. With application of the ertapenem CLSI clinical breakpoint for resistance (MIC ≥1 mg/L), only six isolates (0.2%) were ertapenem-resistant, and only three of these were available for molecular characterization. Of those three, only one was ESBL-positive (CTX-M-14), and two were carbapenemase-positive (OXA-48). All three were negative for, VIM, NDM and KPC carbapenemases. Although the level of ertapenem resistance in E. coli is very low, further monitoring of ertapenem susceptibility and molecular characterization of ertapenem-resistant isolates is needed.

Sir, Clarithromycin is the most active macrolide against Helicobacter pylori, but pH reduction markedly decreases its activity. This is a cause of concern because H. pylori is found on the gastric mucosa, where the pH is low, c. 5.5. In addition, clarithromycin resistance is increasing in H. pylori, with prevalences attaining at least 10% in several countries. Telithromycin is a new ketolide antibiotic that has been found to be highly active against a variety of microorganisms. We determined its activity, at pH 7.4, 6.5 and 5.9, against 30 macrolide-susceptible and 15 -resistant clinical strains of H. pylori isolated from gastric biopsies in Henri-Mondor hospital between 1997 and 1999. H. pylori ATCC 43504 was used as a control strain. MICs of clarithromycin and telithromycin were determined by the agar dilution technique using Mueller–Hinton agar (Oxoid, Lyon, France) supplemented with 10% fresh horse blood. A Steers inoculating device was used to place 10–10 cfu per spot (2 L per spot) on to the plates (52 spots per plate), which were examined for growth after 72 h of incubation at 37 C under microaerophilic conditions. Strains were considered resistant to clarithromycin if the MIC was 1 mg/L. At pH 7.4, telithromycin was four-fold less active than clarithromycin against clarithromycin-susceptible strains, the MIC50 of telithromycin being 0.125 mg/L (Table). In agreement with previous findings, pH reduction caused a marked increase in the MICs of clarithromycin for clarithromycin-susceptible strains: at pH 6.5, the MIC50 and MIC90 increased twoand eight-fold, respectively; at pH 5.9, eightand 16-fold increases were observed (Table). The activity of telithromycin against clarithromycinsusceptible strains was similarly affected by pH reduction (Table). None the less, the MICs of telithromycin at pH 5.9 for most strains remained below the maximum achievable serum concentration of telithromycin: 2.27 mg/L after 800 mg od for 7 days. The A2142G and A2143G mutations in the 23S rRNA gene that are implicated in clarithromycin resistance of H. pylori were detected by restriction fragment length polymorphism analysis of PCR-amplified DNA, using MboII and BsaI restriction enzymes (New England Biolabs, Beverly, MA, USA), as described previously. All clarithromycin-resistant strains carried either a A2142G (five strains) or A2143G mutation (10 strains). H. pylori possesses two 23S rRNA genes and both were mutated in all strains. In agreement with previous findings, MICs of clarithromycin at pH 7.4 were significantly higher for A2142G mutants (64–128 mg/L) than for A2143G mutants (8–32 mg/L) (Table). Telithromycin was not spared by resistance but the two mutations, and especially the A2142G mutation, were associated with a lower increase in the MICs of telithromycin, MICs for A2142G and A2143G mutants being 8–32 and 4–32 mg/L, respectively (Table). Macrolides and ketolides interact with bacterial 23S rRNA making contacts with the peptidyl transferase loop in domain V but also with the hairpin 35 in domain II. A2142 and A2143 are included in this loop and mutations such as those observed in H. pylori, as well as methylation of A2142 (the principal resistance mechanism in Grampositive bacteria but which has not been described in H. pylori) markedly decrease the affinity of both classes of drugs for the loop. However, while interactions of macrolides and ketolides with domain V are similar, their interactions with domain II differ. The stronger interaction of ketolides with hairpin 35 could explain their good activity against strains that contain ribosomes with methylated rRNA. This could also explain why ketolides are less affected by rRNA gene mutations in H. pylori. In the future, it may be possible to discover new ketolides whose interaction with hairpin 35 is sufficient for the binding of the antibiotic, so that these drugs could remain active against macrolide-resistant rRNA mutants of H. pylori. Correspondence Journal of Antimicrobial Chemotherapy (2001) 48, 735–748