Rapid identification and subsequent contextualization of an outbreak of methicillin-resistant Staphylococcus aureus in a neonatal intensive care unit using nanopore sequencing.

Outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) are well described in the neonatal intensive care unit (NICU) setting. Genomics has revolutionized the investigation of such outbreaks; however, to date, this has largely been completed retrospectively and has typically relied on short-read platforms. In 2022, our laboratory established a prospective genomic surveillance system using Oxford Nanopore Technologies sequencing for rapid outbreak detection. Herein, using this system, we describe the detection and control of an outbreak of sequence-type (ST)97 MRSA in our NICU. The outbreak was identified 13 days after the first MRSA-positive culture and at a point where there were only two known cases. Ward screening rapidly defined the extent of the outbreak, with six other infants found to be colonized. There was minimal transmission once the outbreak had been detected and appropriate infection control measures had been instituted; only two further ST97 cases were detected, along with three unrelated non-ST97 MRSA cases. To contextualize the outbreak, core-genome single-nucleotide variants were identified for phylogenetic analysis after de novo assembly of nanopore data. Comparisons with global (n=45) and national surveillance (n=35) ST97 genomes revealed the stepwise evolution of methicillin resistance within this ST97 subset. A distinct cluster comprising nine of the ten ST97-IVa genomes from the NICU was identified, with strains from 2020 to 2022 national surveillance serving as outgroups to this cluster. One ST97-IVa genome presumed to be part of the outbreak formed an outgroup and was retrospectively excluded. A second phylogeny was created using Illumina sequencing, which considerably reduced the branch lengths of the NICU isolates on the phylogenetic tree. However, the overall tree topology and conclusions were unchanged, with the exception of the NICU outbreak cluster, where differences in branch lengths were observed. This analysis demonstrated the ability of a nanopore-only prospective genomic surveillance system to rapidly identify and contextualize an outbreak of MRSA in a NICU.

Antimicrobial Resistance in Selected Bacteria from Food Animals in New Zealand 2018-2022.

Antimicrobial resistance (AMR) presents a significant threat to human health worldwide. One important source of antimicrobial-resistant infections in humans is exposure to animals or animal products. In a phased survey, we investigated AMR in 300 Escherichia coli isolates and 300 enterococci (Enterococcus faecalis and E. faecium) isolates each from the carcasses of poultry, pigs, very young calves, and dairy cattle (food animals); all Salmonella isolates from poultry, very young calves, and dairy cattle; and 300 Campylobacter (Campylobacter jejuni and C. coli) isolates from poultry. The highest resistance levels in E. coli were found for sulfamethoxazole, tetracycline, and streptomycin, for all food animals. Cefotaxime-resistant E. coli were not found and low resistance to ciprofloxacin, colistin, and gentamicin was observed. The majority of enterococci isolates from all food animals were bacitracin-resistant. Erythromycin- and/or tetracycline-resistant enterococci isolates were found in varying proportions from all food animals. Ampicillin- or vancomycin-resistant enterococci isolates were not identified, and ciprofloxacin-resistant E. faecalis were not found. Salmonella isolates were only recovered from very young calves and all eight isolates were susceptible to all tested antimicrobials. Most Campylobacter isolates were susceptible to all tested antimicrobials, although 16.6% of C. jejuni were resistant to quinolones and tetracycline. Results suggest that AMR in E. coli, enterococci, Salmonella, and Campylobacter isolates from food animals in New Zealand is low, and currently, AMR in food animals poses a limited public health risk. Despite the low prevalence of AMR in this survey, ongoing monitoring of antimicrobial susceptibility in bacteria from food animals is recommended, to ensure timely detection of AMR with potential impacts on animal and human health.

Clostridioides difficile infection in a rural New Zealand secondary care centre: an incidence case-control study.

BACKGROUND: Clostridioides difficile infection (CDI) is a form of antibiotic-associated infectious diarrhoea resulting in significant morbidity and mortality. Community-acquired disease in low-risk individuals is increasingly recognised. There are limited New Zealand data published. AIM: To determine the incidence and location of onset of CDI cases in the Manawatu region, and further describe the demographics, risk factors and prevalent C. difficile ribotypes of the population. METHODS: We performed an incidence case-control study of CDI in the Manawatu region between September 2018 and September 2019. Cases were matched to controls with a negative test for C. difficile. Demographic and comorbidity data, location of onset, drug exposure, disease recurrence and 30-day mortality were collected. Ribotype analysis was performed on C. difficile isolates. RESULTS: Thirty-two specimens tested toxin positive over 12 months, yielding an incidence of 18.3 cases per 100 000 person-years. Twenty-five percent of cases had community onset disease. Cases were more likely to have had amoxicillin/clavulanate or ceftriaxone prescribed. Elevated blood white cell count and lower HbA1c were significantly associated with CDI. The dominant ribotype was 014/020. Two cases were RT 023. CONCLUSION: Our data are similar to previous national data. RT 023 has not been previously reported in New Zealand and has been associated with severe colitis. We demonstrated a significant proportion of community-acquired cases and the true incidence might be higher. Vigilance for community onset disease is required. These data may allow observation of temporal changes in incidence and infection patterns of CDI in New Zealand.

Plasmid-mediated AmpC beta-lactamase-producing Escherichia coli causing urinary tract infection in the Auckland community likely to be resistant to commonly prescribed antimicrobials.

AIM: To estimate the prevalence and characterise plasmid-mediated AmpC beta-lactamase (PMACBL)- producing Escherichia coli in the Auckland community. METHOD: All cefoxitin non-susceptible (NS) E. coli identified at the two Auckland community laboratories between 1 January and 31 August 2011 were referred to ESR for boronic acid double-disc synergy testing, to detect the production of AmpC beta-lactamase, and polymerase chain reaction (PCR) to identify the presence of PMACBL genes. PMACBL-producing isolates were typed using pulsed-field gel electrophoresis (PFGE), and PCR was used to determine their phylogenetic group and to identify multilocus sequence type (ST)131. Antimicrobial susceptibility testing and detection of extended-spectrum beta-lactamases (ESBLs) were performed according to the Clinical and Laboratory Standards Institute recommendations. RESULTS: 101 (51%) and 74 (37%) of 200 non-duplicate cefoxitin-NS E. coli were PMACBL producers or assumed hyper-producers of chromosomal AmpC beta-lactamase, respectively. The prevalence of PMACBL-producing E. coli was 0.4%. PMACBL-producing E. coli were significantly less susceptible to norfloxacin, trimethoprim and nitrofurantoin than E. coli that produced neither a PMACBL nor an ESBL. Very few (4%) PMACBL-producing E. coli co-produced an ESBL. Most (88%) of the PMACBL-producing isolates had a CMY-2-like PMACBL. The PMACBL-producing E. coli isolates were diverse based on their PFGE profiles, 44% belonged to phylogenetic group D, and only four were ST131. 100 of the 101 PMACBL-producing E. coli were cultured from urine, and were causing urinary tract infection (UTI) in the majority of patients. The median patient age was 56 years and most (94%) of the patients were women. A greater proportion of patients with community-acquired UTI caused by PMACBL-producing E. coli received a beta-lactam antimicrobial than patients with community-acquired UTI caused by other non-AmpC, non-ESBL-producing E. coli. Thirty-six (43%) patients with community-acquired UTI due to PMACBL-producing E. coli were neither hospitalised nor had any antimicrobial treatment in the previous 6 months. CONCLUSION: The prevalence of PMACBL-producing E. coli was relatively low in the Auckland community, but has increased in recent years. Typing revealed that the majority of the PMACBL-producing E. coli in the Auckland region were genetically unrelated meaning that a point source or direct person to person transmission are not drivers of local community spread currently. The isolates were more resistant to non-beta-lactam antimicrobials than other non-AmpC, non-ESBL-producing E. coli, leaving few treatment options. The majority of the PMACBL-producing E. coli isolates seemed to be acquired in the community and were most frequently isolated from women with UTI. A large proportion of patients with community-acquired UTI had not been hospitalised nor had any antimicrobial treatment in the previous 6 months.

Severe Clostridium difficile infection in New Zealand associated with an emerging strain, PCR-ribotype 244.

AIM: To compare disease severity and clinical outcome of Clostridium difficile infection (CDI) due to PCR-ribotype (RT) 244 with CDI due to other strains present in Auckland. METHOD: A retrospective, case-control study was conducted. Ten cases with CDI due to RT 244 were compared with 20 controls infected with other C. difficile strains. RT 244 isolates were further analysed for antimicrobial susceptibility, binary toxin genes and mutations in the tcdC gene. RESULTS: Cases were significantly more likely to have severe disease than controls (OR 9.33; p=0.015). 50% of cases had community-associated CDI compared with 15% of controls (p=0.078). All RT 244 isolates produced binary toxin and had a single-base pair deletion in tcdC at position 117. CONCLUSION: C. difficile RT 244 is a newly recognised strain in New Zealand. It shares several features that characterise RT 027. Given its propensity to cause severe community-associated disease, a heightened awareness of this strain is needed to ensure early testing in patients admitted from the community with identified risk factors for CDI.

In vitro activities of telithromycin, linezolid, and quinupristin-dalfopristin against Streptococcus pneumoniae with macrolide resistance due to ribosomal mutations.

To date, 86 of 7,746 macrolide-resistant Streptococcus pneumoniae isolates from 1999 to 2002 PROTEKT (Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin) surveillance studies were negative for methylase and efflux mechanisms. Mutations in 23S rRNA or the genes encoding riboprotein L4 or L22 were found in 77 of 86 isolates. Six isolates were resistant to quinupristin-dalfopristin and two were resistant to linezolid, while telithromycin demonstrated good activities against all isolates.

Molecular epidemiology of multiresistant Streptococcus pneumoniae with both erm(B)- and mef(A)-mediated macrolide resistance.

Of a total of 1043 macrolide-resistant Streptococcus pneumoniae isolates collected from 24 countries as part of PROTEKT 1999-2000, 71 isolates tested positive for both the mef(A) and erm(B) genes. Of 69 isolates subjected to further molecular investigations, all were resistant to tetracycline, 63 (91.3%) were resistant to penicillin, and 57 (82.6%) were resistant to trimethoprim-sulfamethoxazole. One isolate was also fluoroquinolone resistant, and another was resistant to quinupristin-dalfopristin. The ketolide telithromycin retained activity against all of the isolates. Of the 69 of these 71 isolates viable for further testing, 46 were from South Korea, 13 were from the United States, 8 came from Japan, and 1 each came from Mexico and Hungary. One major clonal complex (59 [85.5%] of 69 isolates) was identified by serotyping (with 85.5% of the isolates being 19A or 19F), pulsed-field gel electrophoresis, and multilocus sequence typing. The remaining isolates were less clonal in nature. Representative isolates were shown to carry the mobile genetic elements Tn1545 and mega, were negative for Tn1207.1, had tetracycline resistance mediated by tet(M), and contained the mef(E) variant of mef(A). All isolates were positive for mel, a homologue of the msr(A) efflux gene. These clones are obviously very efficient at global dissemination, and hence it will be very important to monitor their progress through continued surveillance. Telithromycin demonstrated high levels of activity (MIC for 90% of the strains tested, 0.5 micro g/ml; MIC range, 0.06 to 1 micro g/ml) against all isolates.

Molecular characterization and antimicrobial susceptibility of fluoroquinolone-resistant or -susceptible Streptococcus pneumoniae from Hong Kong.

Fluoroquinolone resistance in Streptococcus pneumoniae isolated from Hong Kong as part of Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin 1999/2000 was found to be due to the spread of the Spain(23F)-1 clone (mainly a Spain(23F)-1-14 variant). All the isolates were multidrug resistant but were susceptible to quinupristin-dalfopristin, linezolid, and telithromycin. The Spain(23F)-1 clone also occurred among antimicrobial-susceptible isolates, which suggests that the primary source of this clone may have been Asia rather than Iberia.