High Prevalence and Mechanism Associated With Extended Spectrum Beta-Lactamase-Positive Phenotype in Laribacter hongkongensis

In this study, we reported the prevalence and mechanism associated with the extended-spectrum beta-lactamase (ESBL)-positive phenotype in Laribacter hongkongensis isolated from patients and fish. Using the inhibition zone enhancement test, 20 (95.2%) of the 21 patient strains and 8 (57.1%) of the 14 fish strains were tested ESBL-positive. However, ESBL genes, including SHV, TEM, CTX-M, GES, and PER, were not detected in all of these 28 L. hongkongensis isolates. No ESBL gene could be detected in either the complete genome of L. hongkongensis HLHK9 or the draft genome of PW3643. PCR and DNA sequencing revealed that all the 35 L. hongkongensis isolates (showing both ESBL-positive and ESBL-negative phenotypes) were positive for the ampC gene. When the AmpC deletion mutant, HLHK9ΔampC, was subject to the zone enhancement test, the difference of zone size between ceftazidime/clavulanate and ceftazidime was less than 5 mm. When boronic acid was added to the antibiotic disks, none of the 28 “ESBL-positive” isolates showed a ≥ 5 mm enhancement of inhibition zone size diameter between ceftazidime/clavulanate and ceftazidime and between cefotaxime/clavulanate and cefotaxime. A high prevalence (80%) of ESBL-positive phenotype is present in L. hongkongensis. Overall, our results suggested that the ESBL-positive phenotype in L. hongkongensis results from the expression of the intrinsic AmpC beta-lactamase. Confirmatory tests should be performed before issuing laboratory reports for L. hongkongensis isolates that are tested ESBL-positive by disk diffusion clavulanate inhibition test.

Occurrence and diversity of CRISPRs in Laribacter hongkongensis isolates from animals, environment and diarrhea patients in southern China

Background Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins are functional elements of archaea and bacteria, and they form the genetic barrier that reduces the transformation of horizontal genes by an unknown mechanism. Results we searched for CRISPRs in 118 Laribacter hongkongensis strains isolated from patients, animals, and water reservoirs. Two CRISPR loci, designated CRISPR4.1 and CRISPR4.2, were identified in L. hongkongensis strains. A CRISPR4.1/cas system was detected in 91.5% (108/118) of the isolates and belonged to the I-F/Ypest subtype of CRISPR/cas systems, while the remaining ten strains only possessed cas genes without the CRISPR4.1 array. The CRISPR4.2 locus was an orphan locus and existed in 72.0% (85/118) L. hongkongensis strains. Meanwhile, a total of 2562 spacers and 980 unique spacers, arranged in 77 alleles, were found, including 1613 (579 unique, 40 alleles) for CRISPR4.1 and 949 (401 unique, 37 alleles) for CRISPR4.2. The results showed that limited spacers had matches in the plasmid (34), phage (19) and bacteria chromosomal sequences (4) from GenBank databases. Consequently, we found the diversity and activity of CRISPRs from human and frog isolates were closer and higher, respectively, than those of the fish isolates. Conclusions CRISPR4.1 and CRISPR4.2 exist extensively in L. hongkongensis and belong to the I-F/Ypest subtype. Our results indicate that the CRISPR4.1/cas system is a functional unit, and CRISPR4.2 is perhaps a degenerated and nonfunctional locus. Considering that the CRISPR4.1 locus is relatively active, diverse, and conserved at different levels, the biological functions, types, and epidemiological characteristics of CRISPR loci may be worth studying.