Ceftazidime-avibactam resistance and restoration of carbapenem susceptibility in KPC-producing Klebsiella pneumoniae infections: A case series.

OBJECTIVES: Since the introduction of the ß-lactam/ß-lactamase inhibitor ceftazidime-avibactam (CZA), rapid evolution of resistance has been reported in different KPC-producing Klebsiella pneumoniae isolates. In this multicenter retrospective study, we describe the emergence of CZA resistance and evaluate the mutations that might be responsible for the restoration of carbapenem susceptibility. METHODS: During a study period of 18 months, KPC-producing K. pneumoniae isolates of five hospitalized patients were collected with phenotypic development of CZA resistance. RESULTS: In vitro restoration of carbapenem susceptibility during treatment was observed in 3 isolates. Whole genome sequencing of these isolates showed a D179Y mutation in the KPC gene of 2 variants and a KPC-2 with a Δ242-GT-243 deletion (KPC-14). Two KPC-3 variants showed CZA resistance with sustained carbapenemase activity without genomic adaptations in the KPC gene. CONCLUSIONS: This study confirms the emergence of CZA resistance in KPC K. pneumoniae. The role of carbapenems in treating patients with these variants is unclear and combination therapies warrant further investigation.

Direct detection of extended-spectrum beta-lactamases (CTX-M) from blood cultures by LC-MS/MS bottom-up proteomics.

Rapid bacterial species identification and antibiotic susceptibility testing in positive blood cultures have an important impact on the antibiotic treatment for patients. To identify extended-spectrum beta-lactamases (ESBL) directly in positive blood culture bottles, we developed a workflow of saponin extraction followed by a bottom-up proteomics approach using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The workflow was applied to positive blood cultures with Escherichia coli and Klebsiella pneumoniae collected prospectively in two academic hospitals over a 4-month period. Of 170 positive blood cultures, 22 (12.9%) contained ESBL-positive isolates based on standard susceptibility testing. Proteomic analysis identified CTX-M ESBLs in 95% of these isolates directly in positive blood cultures, whereas no false positives were found in the non-ESBL producing positive blood cultures. The results were confirmed by molecular characterisation of beta-lactamase genes. Based on this proof-of-concept study, we conclude that LC-MS/MS-based protein analysis can directly identify extended-spectrum beta lactamases in E. coli and K. pneumoniae positive blood cultures, and could be further developed for application in routine diagnostics.

Implications of ad-hoc molecular typing for infection control measures in a multi-cluster, multi-phenotypic Serratia marcescens outbreak in a neonatal intensive care unit.

BACKGROUND: Serratia marcescens is known to cause outbreaks in neonatal intensive care units (NICUs). Traditionally epidemiological data, antimicrobial resistance patterns and epidemiological typing have been used to guide infection prevention methods. Whole-genome sequencing (WGS) applications such as core-genome multi-locus sequence typing (cgMLST) applied during an outbreak would potentially yield more information. AIM: To use cgMLST to acquire detailed information on the source and spread of bacteria, enabling more efficient control measures during an S. marcescens outbreak at a NICU. METHODS: Neonates admitted to the NICU of the Leiden University Medical Center (LUMC) during an outbreak between September 2023 and January 2024, with S. marcescens being cultured, were included. Environmental samples were taken to search for a common source, antibiotic susceptibility testing was performed, and antimicrobial resistance genes were analysed. FINDINGS: S. marcescens strains from 17 of the 20 positive patients were available for molecular typing. The cgMLST scheme revealed five different complex types consisting of four separate clusters. Multiple clusters made an unidentified persistent environmental source as cause of the outbreak less likely, leading to a quick downscaling of infection prevention measures. Differences were shown in aminoglycoside resistance patterns of isolates within the same complex types and patients. CONCLUSION: The use of ad-hoc cgMLST provided timely data for rational decision-making during an S. marcescens outbreak at the NICU. Antibiotic phenotyping alone was found not to be suitable for studying clonal spread during this outbreak with S. marcescens.

Characterization of fluoroquinolone and cephalosporin resistance mechanisms in Enterobacteriaceae isolated in a Dutch teaching hospital reveals the presence of an Escherichia coli ST131 clone with a specific mutation in parE.

OBJECTIVES: To characterize the mechanisms of fluoroquinolone and cephalosporin resistance in Enterobacteriaceae from a Dutch teaching hospital in 2008. METHODS: We sequenced gyrA, gyrB, parC and parE. The presence of plasmid-encoded genes qnrA, qnrB, qnrS, aac(6')-Ib, qepA, bla(TEM), bla(SHV,) bla(OXA), bla(CTX-M) and bla(AmpC) was studied by PCR. Escherichia coli isolates were further characterized by AFLP and multilocus sequence typing (MLST). RESULTS: In total, 49 E. coli, 16 Klebsiella pneumoniae and 3 Enterobacter cloacae isolates were investigated. Mutations in gyrA were found in all E. coli isolates. Forty-five (92%) E. coli isolates carried at least one point mutation in parC. Most E. coli isolates (59%) also carried mutations in parE, of which I529L was the most prevalent. I529L was unequivocally associated with E. coli sequence type (ST) 131. This single-nucleotide polymorphism (SNP) was later also found in eight out of nine ST131 strains from another collection. Twenty-nine E. coli isolates carried extended-spectrum ß-lactamase (ESBL) genes, predominantly bla(CTX-M-15). In E. coli, aac(6')-Ib-cr was the predominant plasmid-mediated resistance mechanism, whereas in K. pneumoniae qnr genes were found mostly. In K. pneumoniae isolates, qnr and aac(6')-Ib-cr co-occurred with ESBL genes (n = 13; bla(CTX-M) and bla(SHV)) and/or bla(AmpC) (n = 3; bla(DHA-1)). CONCLUSIONS: E. coli ST131 was the predominant clone, which accumulated a high number of chromosomal mutations. The I529L SNP in parE was a signature of most, but not all, ST131 strains. In contrast to E. coli, fluoroquinolone resistance mechanisms were predominantly plasmid-encoded in K. pneumoniae.

Borderline oxacillin-resistant Staphylococcus aureus carriage among healthcare workers at neonatal intensive care unit and paediatric ward.

BACKGROUND: During a meticillin-resistant Staphylococcus aureus contact tracing and screening investigation, two borderline oxacillin-resistant Staphylococcus aureus (BORSA)-positive screening cultures were encountered among neonatal intensive care unit (NICU) healthcare workers (HCWs). This finding led to further investigations. AIM: To assess the likelihood of an outbreak with direct transmission among HCWs. METHODS: An infection control team was initiated after the discovery. The team initiated additional infection control measures and evaluated new findings. All NICUs and paediatric ward HCWs were screened for BORSA carriage, and a prospective BORSA seven-week monitoring period for patients was observed. To assess the likelihood of an outbreak with direct transmission among HCWs, the BORSA isolates were analysed using augmented fragment length polymorphism and whole-genome sequencing (WGS). FINDINGS: Positive HCWs were prohibited from clinical work while awaiting the results from the screening programme. In all, 127 NICU and 77 general paediatric ward HCWs were screened for BORSA carriage; five HCWs were BORSA positive. Seventy-two patients were screened during the seven-week period yielding a total of 138 cultures, ranging from one to nine cultures per patient. No spread from HCWs to patients occurred, and the BORSA screening programme was discontinued. WGS analysis with core genome multi-locus sequence typing of all five BORSA strains showed relatedness between two NICU strains. CONCLUSION: During a seven-week period, no transmission from BORSA-positive HCWs to neonates was observed in either screening or clinical cultures. More vigilance and experience is needed to design adequate evidence-based interventions in the future for this vulnerable population.

Spread of ESBL-producing Escherichia coli in nursing home residents in Ireland and the Netherlands may reflect infrastructural differences.

A prevalence study in two nursing homes (one each in the Netherlands and Ireland) found four (11%) Dutch and six (9%) Irish residents colonized with 11 extended-spectrum beta-lactamase-producing Escherichia coli, 10 of which contained CTX-M-15. Four Dutch isolates, from three residents of the same ward, belonged to E. coli O25:H4, sequence type (ST) 131 and were part of the same cluster type by whole-genome sequencing. Four Irish residents on three different wards were colonized with an identical E. coli O89:H9, ST131, complex type 1478. Cross-transmission between three Irish wards may reflect differences in nursing home infrastructure, specifically communal areas and multi-bedded resident rooms.

Dissemination of Bacillus cereus in a paediatric intensive care unit traced to insufficient disinfection of reusable ventilator air-flow sensors.

Over a two-week period in November 2006, vancomycin-resistant Bacillus cereus was isolated from respiratory samples from six ventilated paediatric intensive care unit (PICU) patients. To investigate the possibility of a common source and extent of the dissemination, all procedures related to mechanical ventilation were monitored and surveillance cultures performed. B. cereus was isolated from reusable air-flow sensors, before and after on-site disinfection with 70% alcohol. The organism was also isolated from respiratory samples from three other ventilated patients and from two ventilation grids in the ceiling of PICU, as well as from the alcohol solution itself. Using amplified fragment length polymorphism (AFLP) typing, B. cereus strains from the six PICU patients together with isolates recovered from the air-flow sensors and the alcohol solution were shown to be closely related. Isolates from the ventilation grids demonstrated different AFLP patterns to the outbreak strain. Intervening measures, including disinfection by autoclaving all reusable air-flow-guiding parts and the use of disposable non-autoclavable parts, resulted in rapid termination of the outbreak. B. cereus infections can cause significant morbidity, particularly in intensive care patients. Disinfection of all air-flow-guiding reusable parts for mechanical ventilation should be addressed with great care and should include effective autoclaving in order to eradicate spores.