Prevalence,phenotypic and genetic characteristics of prolyliminopeptidase‐negative Neisseria gonorrhoeae isolates in Sweden during 2000–2007

Johansson E, Fredlund H, Unemo M. Prevalence, phenotypic and genetic characteristics of prolyliminopeptidase‐negative Neisseria gonorrhoeae isolates in Sweden during 2000–2007. APMIS 2009; 117: 900–4. In Neisseria gonorrhoeae culture diagnostics, species confirmation is commonly performed using commercial biochemical tests relying on prolyliminopeptidase (PIP) activity. It was previously shown that one PIP‐negative strain was mainly globally transmitted during 2000–2004. The aims were to investigate the prevalence and phenotypic and genetic characteristics of PIP‐negative N. gonorrhoeae isolates in Sweden during 2000–2007. Gonococcal isolates (n = 1230) cultured in Sweden during 2000–2007 were characterized using PIP screening, antibiogram, serovar determination, pip and porB gene sequencing, and N. gonorrhoeae multiantigen sequence typing (NG‐MAST). Fifteen (1.2%) of the isolates were PIP‐negative. Of those, 13 (87%) were indistinguishable to the previously described globally transmitted strain, i.e. displayed serovar IB‐4 (Bpyvut), similar antibiograms, ST210 (n = 10)/ST292 (n = 3) and contained an identical single nucleotide pip gene deletion. Wherever high reliance is placed on PIP activity for N. gonorrhoeae species confirmation, changes in the diagnostic strategies may need to be considered and/or monitoring of the PIP activity is crucial.     

Phenotypic and genotypic properties of <Emphasis Type="Italic">Neisseria gonorrhoeae</Emphasis> isolates in Norway in 2009: antimicrobial resistance warrants an immediate change in national management guidelines

Despite rapidly diminishing treatment options for Neisseria gonorrhoeae and high levels of ciprofloxacin resistance worldwide, Norwegian guidelines still recommend ciprofloxacin as empirical treatment for gonorrhea. The present study aimed to characterize phenotypical and genotypical properties of N. gonorrhoeae isolates in Norway in 2009. All viable N. gonorrhoeae isolates (n = 114) from six university hospitals in Norway (2009) were collected, representing 42% of all notified gonorrhea cases. Epidemiological data were collected from the Norwegian Surveillance System for Communicable Diseases and linked to phenotypical and genotypical characteristics for each N. gonorrhoeae isolate. Resistance levels to the antimicrobials examined were: ciprofloxacin 78%, azithromycin 11%, cefixime 3.5%, ceftriaxone 1.8%, and spectinomycin 0%. The minimum inhibitory concentrations of gentamicin varied from 1.5 to 8 mg/L. Forty-one (36%) of the isolates were β-lactamase-producing, 17 displayed penA mosaic alleles, and 72 different N. gonorrhoeae multiantigen sequence types (ST; 37 novel) were identified. The most common ST was ST1407 (n = 11), containing penA mosaic allele. Four of these isolates displayed intermediate susceptibility/resistance to cefixime. The N. gonorrhoeae strains circulating in Norway were highly diverse. The level of ciprofloxacin resistance was high and the Norwegian management guidelines should promptly exclude ciprofloxacin as an empirical treatment option for gonorrhea.     

Antimicrobial resistance and molecular epidemiology using whole-genome sequencing of <Emphasis Type="Italic">Neisseria gonorrhoeae</Emphasis> in Ireland, 2014–2016: focus on extended-spectrum cephalosporins and azithromycin

High-level resistance and treatment failures with ceftriaxone and azithromycin, the first-line agents for gonorrhoea treatment are reported and antimicrobial-resistant Neisseria gonorrhoeae is an urgent public health threat. Our aims were to determine antimicrobial resistance rates, resistance determinants and phylogeny of N. gonorrhoeae in Ireland, 2014–2016. Overall, 609 isolates from four University Hospitals were tested for susceptibility to extended-spectrum cephalosporins (ESCs) and azithromycin by the MIC Test Strips. Forty-three isolates were whole-genome sequenced based on elevated MICs. The resistance rate to ceftriaxone, cefixime, cefotaxime and azithromycin was 0, 1, 2.1 and 19%, respectively. Seven high-level azithromycin-resistant (HLAzi-R) isolates were identified, all susceptible to ceftriaxone. Mosaic penA alleles XXXIV, X and non-mosaic XIII, and G120K plus A121N/D/G (PorB1b), H105Y (MtrR) and A deletion (mtrR promoter) mutations, were associated with elevated ESC MICs. A2059G and C2611T mutations in 23S rRNA were associated with HLAzi-R and azithromycin MICs of 4–32 mg/L, respectively. The 43 whole-genome sequenced isolates belonged to 31 NG-MAST STs. All HLAzi-R isolates belonged to MLST ST1580 and some clonal clustering was observed; however, the isolates differed significantly from the published HLAzi-R isolates from the ongoing UK outbreak. There is good correlation between previously described genetic antimicrobial resistance determinants and phenotypic susceptibility categories for ESCs and azithromycin in N. gonorrhoeae. This work highlights the advantages and potential of whole-genome sequencing to be applied at scale in the surveillance of antibiotic resistant strains of N. gonorrhoeae, both locally and internationally.     

Strain Typing and Antimicrobial Resistance of Fluoroquinolone-Resistant Neisseria gonorrhoeae Causing a California Infection Outbreak

Antimicrobial-resistant Neisseria gonorrhoeae is an emerging public health problem as a result of the alarming limitation in treatment options. We examined an outbreak in California of fluoroquinolone-resistant Neisseria gonorrhoeae (QRNG) by evaluation of a combination of routine isolates from the Gonococcal Isolate Surveillance Project and isolates collected by expanded surveillance performed between April 2000 and June 2002. QRNG isolates were characterized by two methods: (i) determination of a combination of antibiogram, auxotype, serovar, Lip type, and patterns of amino acid alteration in the quinolone resistance-determining region of GyrA and ParC (ASLGP) and (ii) pulsed-field gel electrophoresis (PFGE). Strain typing was used to describe the QRNG outbreak strains and the associated antimicrobial resistance profiles. Among 79 isolates that were completely characterized, we identified 20 different ASLGP strain types, and 2 of the types were considered to belong to outbreak strains that comprised 65% (51/79) of the isolates. By PFGE typing, there were 24 different strain types, and 4 of these were considered outbreak types and comprised 66% (52/79) of the isolates. The overall agreement between the typing methods in distinguishing outbreak strains and non-outbreak strains was 84% (66/79). The most common QRNG ASLGP strain type had chromosomally mediated resistance to penicillin and tetracycline and an azithromycin MIC of 0.5 μg/ml. The occurrence of an outbreak caused by QRNG strains that could fail to be eradicated by most antibiotic classes reinforces the serious problem with antimicrobial resistance in Neisseria gonorrhoeae that the public health system faces. Adherence to a regimen with the recommended antibiotics at the appropriate dose is critical, and monitoring for antimicrobial susceptibility needs to be actively maintained to adapt treatment guidelines appropriately.Neisseria gonorrhoeae antimicrobial susceptibility in the United States has been monitored by the Gonococcal Isolate Surveillance Project (GISP) at the Centers for Disease Control and Prevention (CDC), Atlanta, GA, since 1986. The need for routine surveillance for antimicrobial susceptibility was established with the emergence of chromosomal resistance to penicillin and tetracycline in Neisseria gonorrhoeae in the United States in the 1970s and 1980s (32). With the development of plasmid-mediated resistance to penicillin and tetracycline, the treatment recommendations were changed to ceftriaxone, with cefixime and ciprofloxacin being oral alternatives (29). Fluoroquinolone-resistant Neisseria gonorrhoeae (QRNG) was first isolated in Hawaii in 1991, but it was not until after 1998 that the frequency of QRNG started to escalate (5). In California, the frequency of QRNG isolates increased from less than 1% in 2000 to 25.4% in 2005 (2). At the onset of the occurrence of QRNG in California, expanded surveillance suggested an outbreak among men who have sex with men (MSM) in Southern California (1). A transmission network was constructed from the epidemiological connections determined from the sexual partnerships and venues where sexual partners met and could be supported by strain typing of the isolates from these cases among MSM (18). In this paper, we report on the complete strain typing and examine the antimicrobial resistance profiles among the isolates identified in this surveillance. This should provide important information on the origins of evolving Neisseria gonorrhoeae antimicrobial resistance in the United States by characterizing the strains that became established on the continent.     

Epidemiology and antimicrobial resistance profile of Neisseria gonorrhoeae in Catalonia,Spain, 2016–2019

Antimicrobial resistance data for Neisseria gonorrhoeae is globally sparse and resistant strains are emerging in Catalonia. We aim to describe epidemiological and antimicrobial resistance in all patients infected with N. gonorrhoeae during the period from 2016 to 2019, using available antimicrobial susceptibility data. We retrospectively analysed confirmed N. gonorrhoeae cases notified to Catalonia’s microbiological reporting system. Antibiotic susceptibility testing (azithromycin, cefixime, ceftriaxone, ciprofloxacin, penicillin, spectinomycin, and tetracycline) was assessed using clinical breakpoints published by the European Committee on Antimicrobial Susceptibility Testing. Incidence rates were calculated and proportions were compared using the χ² test or Fisher’s exact test, and analysed using the Statistical Package for Social Sciences (SPSS 18.0). A total of 14,251 confirmed cases of N. gonorrhoeae were notified. Incidence increased from 30.7 cases/100,000 person-years (p < 0.001) in 2016 to 64.7 in 2019. Culture was available in 6,292 isolates (44.2%), of which 5,377 (85.5%) were resistant to at least one of the antibiotics tested. Azithromycin resistance rose from 6.1% in 2016 to 16% in 2019 (p < 0.001). Only 1.0% (45 cases) were resistant to ceftriaxone. Multidrug-resistant N. gonorrhoeae increased from 0.25% in 2016 to 0.42% in 2019 (p = 0.521). One case presented extensively drug-resistant N. gonorrhoeae. In Catalonia, 10% of the N. gonorrhoeae isolates were resistant to azithromycin in the 2016–2019 period. According to World Health Organization guidelines, resistance above 5% indicates an alert to review treatment guidelines. Antimicrobial susceptibility testing in clinical practice followed by surveillance and interventions are essential to monitor trends and prevent the spread of antimicrobial resistance.

     

A new confirmatory <Emphasis Type="Italic">Neisseria gonorrhoeae</Emphasis> real-time PCR assay targeting the porA pseudogene

The Roche Cobas Amplicor system is widely used for the detection of Neisseria gonorrhoeae but is known to cross react with some commensal Neisseria spp. Therefore, a confirmatory test is required. The most common target for confirmatory tests is the cppB gene of N. gonorrhoeae. However, the cppB gene is also present in other Neisseria spp. and is absent in some N. gonorrhoeae isolates. As a result, laboratories targeting this gene run the risk of obtaining both false-positive and false-negative results. In the study presented here, a newly developed N. gonorrhoeae LightCycler assay (NGpapLC) targeting the N. gonorrhoeae porA pseudogene was tested. The NGpapLC assay was used to test 282 clinical samples, and the results were compared to those obtained using a testing algorithm combining the Cobas Amplicor System (Roche Diagnostics, Sydney, Australia) and an in-house LightCycler assay targeting the cppB gene (cppB-LC). In addition, the specificity of the NGpapLC assay was investigated by testing a broad panel of bacteria including isolates of several Neisseria spp. The NGpapLC assay proved to have comparable clinical sensitivity to the cppB-LC assay. In addition, testing of the bacterial panel showed the NGpapLC assay to be highly specific for N. gonorrhoeae DNA. The results of this study show the NGpapLC assay is a suitable alternative to the cppB-LC assay for confirmation of N. gonorrhoeae-positive results obtained with Cobas Amplicor.     

RFLP analysis of recent Northern Ireland isolates of infectious laryngotracheitis virus: Comparison with vaccine virus and field isolates from England,Scotland and the Republic of Ireland

Restriction fragment length polymorphism (RFLP) analysis was used to assist epidemiological investigations following the recent introduction of infectious laryngotracheitis virus (ILTV) to commercial poultry flocks in Northern Ireland (NI). A 4.9 kbp PCR product of the ILTV ICP4 gene was generated from each of 16 field isolates of ILTV originating from England, Scotland, NI and the Republic of Ireland (RoI) and of the single vaccine strain currently licenced for use within the United Kingdom. With the exception of isolate PV6/94 from RoI, all field isolates generated RFLP patterns, following digestion with HaeIII, similar to that obtained using the vaccinal strain. Following MspI digestion, NI isolates were indistinguishable from the vaccinal strain and recent English isolates. However, one English and one Scottish isolate, both made prior to the introduction of vaccination, and two isolates from RoI generated a second pattern following digestion with MspI.     

Molecular epidemiology of a large community-based outbreak of hepatitis B in Bristol, UK

Background

A large outbreak of hepatitis B virus (HBV) infection in the UK occurred between 2001 and 2005 in Bristol, UK.

Objectives

To identify HBV strains circulating amongst risk groups in the HBV outbreak cohort.

Study design

Cross-sectional study of acute HBV outbreak cases in Bristol.

Results

HBV sequences from sera of 95 of the 237 cases (40%) were characterised. The majority of cases (77%) were found to carry an HBV variant belonging to genotype D, designated HBV^BV. Eighty-eight percent (36/41) of sequences from injection drug users were HBV^BV as were 70% (19/27) from those with heterosexual intercourse as the primary identified risk factor. Of 15 sequences characterised from cases of pre-outbreak acute or chronic hepatitis B residing in Bristol, 40% also carried HBV^BV; the earliest was from a case identified in 1994.

Conclusion

The findings from this study link the spread of HBV^BV from injecting drug users to the general population through heterosexual intercourse during the outbreak. The molecular sequencing of specimens from this outbreak reports the emergence of HBV^BV, a HBV strain circulating in Bristol and South West England, as the cause of one of the largest outbreaks of acute hepatitis B in the UK.     

Mannose‐binding lectin is present in human semen and modulates cellular adhesion of Neisseria gonorrhoeae in vitro

Mannose‐binding lectin (MBL) is an innate immune molecule present in blood and some mucosal tissues, which can influence microbial attachment and inflammatory responses of host cells during infection. In this study MBL was found to be present at a low concentration in semen samples in the range 1·2–24·9 ng/ml. Co‐incubation of bacteria with semen resulted in the binding of MBL to the bacterial surface. Neisseria gonorrhoeae is a common cause of genitourinary infection. MBL bound to N. gonorrhoeae with strain‐to‐strain variation in the intensity of binding and nature of the bacterial receptor. Pretreatment with MBL concentrations similar to those found in human serum modulated the adhesion of N. gonorrhoeae strain FA1090 but not strain MS11 to epithelial cells. This effect was dose‐dependent. This work demonstrates that MBL is present in human semen and modifies cellular responses to N. gonorrhoeae in a concentration‐dependent manner.     

Antigenic similarities in lipopolysaccharides of Haemophilus and Neisseria and expression of a digalactoside structure also present on human cells

Monoclonal antibodies raised against Haemophilus influenzae and Neisseria gonorrhoeae were used to investigate similarities or differences in the lipopolysaccharide antigens of pathogenic and commensal strains of several Gram-negative bacteria indigenous to mucosal surfaces of humans. In immunoblotting experiments, 20 of 36 monoclonal antibodies showed cross-reactions between species of Neisseria and Haemophilus. The common epitopes were present on N. gonorrhoeae, N. meningitidis, N. lactamica, H. influenzae including biogroup aegyptius, and occasionally H. parainfluenzae. No other commensal Neisseria or Gram-negative organisms tested reacted with the monoclonal antibodies with one exception; a single strain of pathogenic Escherichia coli was recognised by a N. gonorrhoeae-specific monoclonal antibody. One monoclonal antibody, raised against N. gonorrhoeae lipopolysaccharide, reacted with N. gonorrhoeae (32 of 59 strains), N. meningitidis (9 of 26 strains), H. influenzae (6 of 16 strains). An epitope expressed by H. influenzae and implicated in its virulence was also present on 14 of 59 strains of N. gonorrhoeae and was shown to comprise a digalactoside structure, α-galactosyl-1,4-β-galactose (Galα1, 4Galβ), also found on human cells.     

Novel meningococcal 4CMenB vaccine antigens - prevalence and polymorphisms of the encoding genes in Neisseria gonorrhoeae

The first cross‐protective Neisseria meningitidis vaccine (focus on serogroup B), the protein‐based 4 component meningococcus serogroup B (4CMenB), includes the New Zealand outer membrane vesicle and three main genome‐derived neisserial antigens (GNAs). These GNAs are fHbp (fused to GNA2091), NHBA (fused to GNA1030) and NadA. In this study, the prevalence and polymorphisms of the nucleotide and amino acid sequences of the 4CMenB antigens in a temporally and geographically diverse collection of N. gonorrhoeae isolates (n = 111) were investigated. All the examined GNA genes, except the nadA gene, were present in all gonococcal isolates. However, 25 isolates contained premature stop codons in the fHbp gene and/or the nhba gene, resulting in truncated proteins. Compared with the 4CMenB antigen sequences in reference strain MC58, the gonococcal strains displayed 67.0–95.4% and 60.9–94.9% identity in nucleotide sequence and amino acid sequence, respectively, in the equivalent GNA antigens. The absence of NadA, lack of universal expression of fHbp and NHBA and the uncertainty regarding the surface exposure of fHbp as well as the function of NHBA in N. gonorrhoeae will likely limit the use of the identical 4CMenB antigens in a gonococcal vaccine. However, possible cross‐immunity of 4CMenB with gonococci and expression and function of the equivalent gonococcal GNAs, as well as of more appropriate GNAs for a gonococcal vaccine, need to be further examined.     

Typing of vancomycin-resistant enterococci with MALDI-TOF mass spectrometry in a nosocomial outbreak setting

ObjectivesTo investigate the usefulness of matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS) typing as a first-line epidemiological tool in a nosocomial outbreak of vancomycin-resistant Enterococcus faecium (VREfm).MethodsFifty-five VREfm isolates, previously characterized by whole-genome sequencing (WGS), were included and analysed by MALDI-TOF MS. To take peak reproducibility into account, ethanol/formic acid extraction and other steps of the protocol were conducted in triplicate. Twenty-seven spectra were generated per isolate, and spectra were visually inspected to determine discriminatory peaks. The presence or absence of these was recorded in a peak scheme.ResultsNine discriminatory peaks were identified. A characteristic pattern of these could distinguish between the three major WGS groups: WGS I, WGS II and WGS III. Only one of 38 isolates belonging to WGS I, WGS II or WGS III was misclassified. However, ten of the 17 isolates not belonging to WGS I, II or III displayed peak patterns indistinguishable from those of the outbreak strain.ConclusionsUsing visual inspection of spectra, MALDI-TOF MS typing proved to be useful in differentiating three VREfm outbreak clones from each other. However, as non-outbreak isolates could not be reliably differentiated from outbreak clones, the practical value of this typing method for VREfm outbreak management was limited in our setting.     

Delineating Community Outbreaks of Salmonella enterica Serovar Typhimurium by Use of Whole-Genome Sequencing: Insights into Genomic Variability within an Outbreak

Whole-genome next-generation sequencing (NGS) was used to retrospectively examine 57 isolates from five epidemiologically confirmed community outbreaks (numbered 1 to 5) caused by Salmonella enterica serovar Typhimurium phage type DT170. Most of the human and environmental isolates confirmed epidemiologically to be involved in the outbreaks were either genomically identical or differed by one or two single nucleotide polymorphisms (SNPs), with the exception of those in outbreak 1. The isolates from outbreak 1 differed by up to 12 SNPs, which suggests that the food source of the outbreak was contaminated with more than one strain while each of the other four outbreaks was caused by a single strain. In addition, NGS analysis ruled in isolates that were initially not considered to be linked with the outbreak, which increased the total outbreak size by 107%. The mutation process was modeled by using known mutation rates to derive a cutoff value for the number of SNP difference to determine whether or not a case was part of an outbreak. For an outbreak with less than 1 month of ex vivo/in vivo evolution time, the maximum number of SNP differences between isolates is two or four using the lowest or highest mutation rate, respectively. NGS of S. Typhimurium significantly increases the resolution of investigations of community outbreaks. It can also inform a more targeted public health response by providing important supplementary evidence that cases of disease are or are not associated with food-borne outbreaks of S. Typhimurium.     

Determination of Disk Diffusion and MIC Quality Control Guidelines for Solithromycin,a Novel Fluoroketolide Antibacterial,against Neisseria gonorrhoeae

This solithromycin quality control study was performed to establish quality control (QC) ranges for the N. gonorrhoeae ATCC 49226 control strain for MIC agar dilution testing (AD) and zones by disk diffusion testing (DD). The following ranges were established: AD, 0.03 to 0.25 μg/ml, and DD, 33 to 43 mm. In January 2015, the CLSI Subcommittee on Antimicrobial Susceptibility Testing approved these ranges, which will be important when evaluating solithromycin against clinical isolates of N. gonorrhoeae.     

Genome sequencing and characterization of an extensively drug-resistant sequence type 111 serotype O12 hospital outbreak strain of Pseudomonas aeruginosa

A series of extensively drug-resistant isolates of Pseudomonas aeruginosa from two outbreaks in UK hospitals were characterized by whole genome sequencing (WGS). Although these isolates were resistant to antibiotics other than colistin, we confirmed that they are still sensitive to disinfectants. The sequencing confirmed that isolates in the larger outbreak were serotype O12, and also revealed that they belonged to sequence type ST111, which is a major epidemic strain of P. aeruginosa throughout Europe. As this is the first reported sequence of an ST111 strain, the genome was examined in depth, focusing particularly on antibiotic resistance and potential virulence genes, and on the reported regions of genome plasticity. High degrees of sequence similarity were discovered between outbreak isolates collected from recently infected patients, isolates from sinks, an isolate from the sewer, and a historical isolate, suggesting that the ST111 strain has been endemic in the hospital for many years. The ability to translate easily from outbreak investigation to detailed genome biology by use of the same data demonstrates the flexibility of WGS application in a clinical setting.     

Utility of Specimens Positive for Neisseria gonorrhoeae by the Aptima Combo 2 Assay for Assessment of Strain Diversity and Antibiotic Resistance

In our jurisdiction, the Aptima Combo 2 assay (Gen-Probe, Inc.) is used to detect Neisseria gonorrhoeae from specimens collected at clinics for sexually transmitted infections (STI) and from select community patients. In addition, swabs are also collected for N. gonorrhoeae culture, susceptibility testing, and sequence typing (ST). Since only a small proportion of samples from provincial cases undergo culture, the available trends in antimicrobial susceptibility and predominant strain types may not be representative of all N. gonorrhoeae infections. Due to the limitations facing the use of N. gonorrhoeae culture to understand these trends in the general community, we performed a molecular analysis for markers of cephalosporin resistance and ST determination by using nucleic acid extracts of specimens sent for Aptima testing. Thirty-four samples submitted for both Aptima testing and N. gonorrhoeae culture from the same anatomic location (within 24 h) were included in the study. Sequence type was determined based on the sequence of the por and tbpB genes, and amino acid changes in the PBP 2 protein, encoded by the penA gene, were considered representative for the assessment of antimicrobial susceptibility. Sequence identity of 100% was observed between the sequences obtained from Aptima-analyzed samples and culture samples. Sequencing results showed an association between decreased susceptibility to extended-spectrum cephalosporins (ESC^ds), tbp allele 110, ST 1407, and amino acid changes (G545S, I312M, and V316T) in the PBP 2 protein. Our data, generated based on a few representative genes, suggest that gonococcal samples positive by Aptima testing can be used to determine single nucleotide polymorphisms associated with ESC^ds and the sequence type based on molecular strain typing. Confirmation of these findings may obviate the need for gonorrhea culture in the future.     

Molecular Assay for Detection of Genetic Markers Associated with Decreased Susceptibility to Cephalosporins in Neisseria gonorrhoeae

The incidence of antimicrobial-resistant Neisseria gonorrhoeae continues to rise in Canada; however, antimicrobial resistance data are lacking for approximately 70% of gonorrhea infections that are diagnosed directly from clinical specimens by nucleic acid amplification tests (NAATs). We developed a molecular assay for surveillance use to detect mutations in genes associated with decreased susceptibility to cephalosporins that can be applied to both culture isolates and clinical samples. Real-time PCR assays were developed to detect single nucleotide polymorphisms (SNPs) in ponA, mtrR, penA, porB, and one N. gonorrhoeae-specific marker (porA). We tested the real-time PCR assay with 252 gonococcal isolates, 50 nongonococcal isolates, 24 N. gonorrhoeae-negative NAAT specimens, and 34 N. gonorrhoeae-positive NAAT specimens. Twenty-four of the N. gonorrhoeae-positive NAAT specimens had matched culture isolates. Assay results were confirmed by comparison with whole-genome sequencing data. For 252 N. gonorrhoeae strains, the agreement between the DNA sequence and real-time PCR was 100% for porA, ponA, and penA, 99.6% for mtrR, and 95.2% for porB. The presence of ≥2 SNPs correlated with decreased susceptibility to ceftriaxone (sensitivities of >98%) and cefixime (sensitivities of >96%). Of 24 NAAT specimens with matched cultures, the agreement between the DNA sequence and real-time PCR was 100% for porB, 95.8% for ponA and mtrR, and 91.7% for penA. We demonstrated the utility of a real-time PCR assay for sensitive detection of known markers for the decreased susceptibility to cephalosporins in N. gonorrhoeae. Preliminary results with clinical NAAT specimens were also promising, as they correlated well with bacterial culture results.     

Emergence and control of an outbreak of infections due to Panton-Valentine leukocidin positive,ST22 methicillin-resistant Staphylococcus aureus in a neonatal intensive care unit

Methicillin resistant Staphylococcus aureus (MRSA) infection can cause significant morbidity and mortality in neonates. We investigated a nosocomial MRSA outbreak in a neonatal intensive care unit (NICU), using a novel typing method. Following two fatal cases, in May 2011, a prospective outbreak investigation was conducted, involving neonates, mothers and healthcare workers in a large tertiary NICU in Sydney. MRSA isolates were characterized by antimicrobial susceptibility testing, a multiplex PCR-based reverse line blot (mPCR/RLB) binary typing system and other molecular typing methods. Over 7 months, 14 neonates were colonized with MRSA and six infected: three with superficial lesions and three with life-threatening disease, including the two index cases, who died despite empirical treatment with vancomycin. Isolates from 15 neonates were indistinguishable by RLB typing and identified as a PVL-producing ST22 SCCmec IV MRSA strain, which was resistant to gentamicin and trimethoprim-sulphamethoxazole. The outbreak strain was also isolated from one healthcare worker, one environmental swab and one father, but the source remained obscure. During the same period several different non-multiresistant and multiresistant MRSA strains were isolated from five neonates, five mothers (including two whose infants were colonized with the outbreak strain), one father, three healthcare workers and two environmental swabs. Rapid turnaround time of typing results allowed us to recognize and define the outbreak and implement targeted infection control interventions. PVL-producing ST22 SCCmec IV MRSA appears to be a virulent and highly transmissible pathogen in the NICU, which was difficult to control.     

Outbreak of long-term intravascular catheter-related bacteremia due to Achromobacter xylosoxidans subspecies xylosoxidans in a hemodialysis unit

Achromobacter xylosoxidans is a rare cause of bacteremia. Over a 2-week period, A. xylosoxidans subsp. xylosoxidans was isolated from blood cultures of four hemodialysis patients with long-term intravascular catheters. A culture from one atomizer that contained diluted 2.5% chlorhexidine, which had been used to disinfect the skin, yielded A. xylosoxidans subsp. xylosoxidans. No further cases were diagnosed once the use of this atomizer was discontinued. Five outbreak-related strains from the four patients and the atomizer were tested by pulsed-field gel electrophoresis (PFGE) under XbaI restriction. The isolates from the first three patients and the atomizer had identical PFGE patterns, confirming the atomizer as the source of the outbreak. The strain isolated from the fourth patient had six more bands than the outbreak strain and was considered possibly related to the outbreak strain. All patients were treated with intravenous levofloxacin. The catheter was removed in only one patient. The three patients in whom the catheter was left in place were also treated with antibiotic lock therapy with levofloxacin. All four patients were cured. This is believed to be the first reported outbreak of central venous catheter-related bacteremia due to A. xylosoxidans and the second reported outbreak with this organism associated with chlorhexidine atomizers. The use of diluted chlorhexidine via atomizers can be dangerous for the care of venous catheters and should be called into question. Patients with long-term intravascular catheter-related bacteremia due to this organism can be treated successfully with systemic antimicrobial therapy in addition to antibiotic lock therapy without catheter removal.     

Outbreak ofEscherichia coli O157:H7 infection in a large family

An outbreak of bloody and nonbloody diarrhoea caused byEscherichia coli O157:H7 including one case of haemolytic uraemic syndrome (HUS) and two cases of haemolytic anaemia, in five siblings (aged 2.5 to 11.3 years) and their playmate was investigated. Using sorbitol-MacConkey agar, colony blot hybridisation, and immunomagnetic separation, Shiga toxin 2-producingEscherichia coli O157:H7 was isolated from all children but the HUS patient; however, this patient had high immunoglobulin M antibody titres againstEscherichia coli O157 lipopolysaccharide.Escherichia coli 0157 isolates from all patients were indistinguishable in serotype, virulence properties, and genomic background, indicating that the same strain caused the infections. These data confirm the importance of person-to-person transmission.