Azithromycin resistance in Escherichia coli and Salmonella from food-producing animals and meat in Europe.

OBJECTIVES: To characterize the genetic basis of azithromycin resistance in Escherichia coli and Salmonella collected within the EU harmonized antimicrobial resistance (AMR) surveillance programme in 2014-18 and the Danish AMR surveillance programme in 2016-19. METHODS: WGS data of 1007 E. coli [165 azithromycin resistant (MIC > 16 mg/L)] and 269 Salmonella [29 azithromycin resistant (MIC > 16 mg/L)] were screened for acquired macrolide resistance genes and mutations in rplDV, 23S rRNA and acrB genes using ResFinder v4.0, AMRFinder Plus and custom scripts. Genotype-phenotype concordance was determined for all isolates. Transferability of mef(C)-mph(G)-carrying plasmids was assessed by conjugation experiments. RESULTS: mph(A), mph(B), mef(B), erm(B) and mef(C)-mph(G) were detected in E. coli and Salmonella, whereas erm(C), erm(42), ere(A) and mph(E)-msr(E) were detected in E. coli only. The presence of macrolide resistance genes, alone or in combination, was concordant with the azithromycin-resistant phenotype in 69% of isolates. Distinct mph(A) operon structures were observed in azithromycin-susceptible (n = 50) and -resistant (n = 136) isolates. mef(C)-mph(G) were detected in porcine and bovine E. coli and in porcine Salmonella enterica serovar Derby and Salmonella enterica 1,4, [5],12:i:-, flanked downstream by ISCR2 or TnAs1 and associated with IncIγ and IncFII plasmids. CONCLUSIONS: Diverse azithromycin resistance genes were detected in E. coli and Salmonella from food-producing animals and meat in Europe. Azithromycin resistance genes mef(C)-mph(G) and erm(42) appear to be emerging primarily in porcine E. coli isolates. The identification of distinct mph(A) operon structures in susceptible and resistant isolates increases the predictive power of WGS-based methods for in silico detection of azithromycin resistance in Enterobacterales.

First isolation and genotyping of Bartonella henselae from a cat living with a patient with cat scratch disease in Southeast Europe.

BACKGROUND: The bacterial genus Bartonella is distributed worldwide and poses a public health risk. Cat-scratch disease caused by B. henselae in Croatia was first described in 1957. It is present throughout the country: a survey of serum samples from 268 Croatian patients with lymphadenopathy showed that 37.7% had IgG antibodies. Despite this prevalence, we are unaware of reports of Bartonella culturing from infected humans or cats in Croatia or elsewhere in southeast Europe. CASE PRESENTATION: Here we describe the diagnosis of a 12-year-old child with lymphadenopathy in Croatia with cat-scratch disease based on antibody detection and clinical signs, and the subsequent culturing and genotyping of B.henselae from the cat's blood. The B. henselae isolate was grown on different blood agar plates and its identity was confirmed based on polymerase chain reaction (PCR) amplification of 16S ribosomal deoxyribonucleic acid (16S rDNA) and sequencing. Multi-locus sequence typing (MLST) identified the strain genotype as sequence type 5, commonly found zoonotic B. henselae strain in cats. The child recovered after azithromycin therapy, and B. henselae in the cat was eliminated within three months after doxycycline treatment. CONCLUSIONS: This is, to our knowledge, the first report of B. henselae culturing and MLST-based genotyping from cat's blood in southeast Europe. Our ability to detect B. henselae in blood through culturing but not PCR suggests that the prevalence of infected cats with low bacteremia is very high, suggesting the need to develop faster, more sensitive detection assays.

First Report of Campylobacter jejuni Strains Belonging to ST-21 Clonal Complex Isolated from Human, Poultry and Wild Birds in Croatia: Antimicrobial Resistance and Genetic Distance.

In the era of growing antimicrobial resistance, a threat affecting humans, endangering animals, as well as livelihoods and food security worldwide, we wanted to find possible explanations for its continuous spread from a new perspective. The ubiquity of resistance genes requires a One Health approach to finding the explanations for continuous AMR spread. The natural transformability of Campylobacter jejuni, its high incidence of infections, and emerging resistance worldwide inspired us to choose C. jejuni ST-21CC to be our pathogen for analyzing its contribution and connection to the cycle of AMR dissemination. ST-21CC is known as a generalist among humans and broilers, the most prevalent lineage worldwide, but it is rarely found in wild birds. Emerging in wild birds, genetic relatedness and similar resistance profiles were expected. We analyzed 23 Croatian C. jejuni strains belonging specifically to ST-21CC from humans, broilers, and wild birds. The genomic data obtained through whole genome sequencing and phenotypic susceptibility data of strains were compared. Our findings suggest high fluoroquinolone resistance in ST-21CC strains, with more diverse genetic backgrounds in wild birds. Intriguing were three isolates of ST-822 (from human and storks), sharing a similar genetic fingerprint.

Risk of zoonoses involving slow-growing non-tuberculous mycobacteria: Survey of antimicrobial resistance among strains from domestic and wild animals.

OBJECTIVES: Non-tuberculous mycobacteria are opportunistic pathogens that cause disease mainly in immunocompromised hosts. The present study assessed the prevalence of antibiotic resistance among such mycobacteria from domestic and wild animals in Croatia sampled during several years within a national surveillance program. METHODS: A total of 44 isolates belonging to nine slow-growing species were genotyped and analyzed for susceptibility to 13 antimicrobials often used to treat non-tuberculous mycobacterial infections in humans. RESULTS: Most prevalent resistance was to moxifloxacin (77.3%), doxycycline (76.9%), and rifampicin (76.9%), followed by ciprofloxacin (65.4%), trimethoprim-sulfamethoxazole (65.4%), and linezolid (61.4%). Few isolates were resistant to rifabutin (7.7%) or amikacin (6.8%). None of the isolates was resistant to clarithromycin. Nearly all isolates (86.4%) were resistant to multiple antibiotics. CONCLUSION: Our findings suggest substantial risk that human populations may experience zoonotic infections with non-tuberculous mycobacteria that will be difficult to treat using the current generation of antibiotics. Future work should clarify how resistance emerges in wild populations of non-tuberculous mycobacteria.

Genetic Diversity and Resistome Analysis of Campylobacter lari Isolated from Gulls in Croatia.

Campylobacter lari is a thermotolerant bacterium that sporadically causes gastrointestinal diseases in humans and can be found in wildlife and the environment. C. lari is an understudied species, especially in wild birds such as gulls. Gulls are potentially good carriers of pathogens due to their opportunistic behavior and tendency to gather in large flocks. During winter and their breeding period, 1753 gulls were captured, and cloacal swabs were taken to be tested for the presence of C. lari. From isolated bacteria, the DNA was sequenced, and sequence types (ST) were determined. Sixty-four swabs were positive for C. lari, and from those, forty-three different STs were determined, of which thirty-one were newly described. The whole genome was sequenced for 43 random isolates, and the same isolates were tested for antimicrobial susceptibility using the broth microdilution method to compare them to WGS-derived antimicrobial-resistant isolates. All the tested strains were susceptible to erythromycin, gentamicin, and chloramphenicol, and all were resistant to ciprofloxacin. Resistance to ciprofloxacin was attributed to a gyrA_2 T86V mutation. Genes connected to possible beta-lactam resistance (blaOXA genes) were also detected.

First Report and Characterization of the mcr-1 Positive Multidrug-Resistant Escherichia coli Strain Isolated from Pigs in Croatia.

The emergence and rapid spread of the plasmid-mediated colistin-resistant mcr-1 gene introduced a serious threat to public health. In 2021, a multi-drug resistant, mcr-1 positive Escherichia coli EC1945 strain, was isolated from pig caecal content in Croatia. Antimicrobial susceptibility testing and whole genome sequencing were performed. Bioinformatics tools were used to determine the presence of resistance genes, plasmid Inc groups, serotype, sequence type, virulence factors, and plasmid reconstruction. The isolated strain showed phenotypic and genotypic resistance to nine antimicrobial classes. It was resistant to colistin, gentamicin, ampicillin, cefepime, cefotaxime, ceftazidime, sulfamethoxazole, chloramphenicol, nalidixic acid, and ciprofloxacin. Antimicrobial resistance genes included mcr-1, blaTEM-1B, blaCTX-M-1, aac(3)-IId, aph(3')-Ia, aadA5, sul2, catA1, gyrA (S83L, D87N), and parC (A56T, S80I). The mcr-1 gene was located within the conjugative IncX4 plasmid. IncI1, IncFIB, and IncFII plasmids were also detected. The isolate also harbored 14 virulence genes and was classified as ST744 and O101:H10. ST744 is a member of the ST10 group which includes commensal, extraintestinal pathogenic E. coli isolates that play a crucial role as a reservoir of genes. Further efforts are needed to identify mcr-1-carrying E. coli isolates in Croatia, especially in food-producing animals to identify such gene reservoirs.

Antimicrobial Resistance in Rapidly Growing Nontuberculous Mycobacteria among Domestic and Wild Animals Emphasizing the Zoonotic Potential.

Non-tuberculous mycobacteria (NTM) are opportunistic pathogens capable of causing infections in humans and animals. The aim of this study was to demonstrate the potential role of domestic and wild animals as a reservoir of multiple resistant, rapidly growing NTM strains representing a potential zoonotic threat to humans. A total of 87 animal isolates belonging to 11 rapidly growing species (visible colonies appear within three to seven days) were genotyped and tested for susceptibility to the 15 most commonly used antibiotics in the treatment of such infections in a human clinic. By determining the antimicrobial susceptibility, the most prevalent resistance was found to cephalosporins (>50%), followed by amoxicillin-clavulanate (31.0%), clarithromycin (23.0%), tobramycin (14.9%) and doxycycline (10.3%). Resistance to imipenem, ciprofloxacin, minocycline and linezolid was notably lower (<7.0%). All tested isolates were susceptible to amikacin and moxifloxacin. The most frequent resistance was proved in the most pathogenic species: M. fortuitum, M. neoaurum, M. vaccae and M. porcinum. Meanwhile, other species displayed a higher sensitivity rate. No significant resistance differences between domestic and wild animals were found. The established significant frequency of resistance highlights the significant zoonotic potential posed by circulating rapidly growing NTM strains, which could lead to challenges in the treatment of these infections.

The impact and complete genome characterisation of viruses involved in outbreaks of gastroenteritis in a farrow-to-finish holding.

Viral enteric pathogens continuously burden intensive pig farming, causing gastrointestinal diseases of epidemic and endemic nature. The present study investigated two diarrhoea outbreaks on a large farrow-to-finish holding and subsequent circulation of outbreak-related enteric viruses. These viruses were characterised by whole genome sequencing, and statistical evaluation of the impact on specific production metrics was performed. The results provided evidence that the Porcine epidemic diarrhoea virus-swine enteric coronavirus (PEDV-SeCoV) S gene recombinant strain was responsible for the first outbreak, whilst Rotavirus A (RVA) in a mixed infection with Rotavirus B (RVB) and porcine kobuvirus (PKV) probably caused the second diarrhoea outbreak. Whole genome characterisation revealed a porcine origin of all viruses involved and significant heterogeneity of RVB strain, proposing four novel genotypes and changes in RVB VP1 genotype classification. The statistical evaluation confirmed only a minor disturbance in the number of weaned pigs per sow, with statistical forecasting showing positive trends. A follow-up study corroborated the endemicity of RVA and PKV, in contrast to PEDV-SeCoV. Punctual, comprehensive and timely investigation of diarrhoea outbreaks is a prerequisite for applying adequate pig health and biosecurity management. Calculating such outbreaks' impact on production metrics can potentially shape future decisions on management improvements.

Description of Mycobacterium pinniadriaticum sp. nov., isolated from a noble pen shell (Pinna nobilis) population in Croatia.

Introduction: Shortly before the mass mortality event of the noble pen shell (Pinna nobilis) population in the south-eastern Adriatic coast, two rapidly growing Mycobacterium strains CVI_P3T (DSM 114013 T, ATCC TSD-295 T) and CVI_P4 were obtained from the organs of individual mollusks during the regular health status monitoring. Methods: The strains were identified as members of the genus Mycobacterium using basic phenotypic characteristics, genus-specific PCR assays targeting the hsp65 and 16S rRNA genes and the commercial hybridization kit GenoType Mycobacterium CM (Hain Lifescience, Germany). MALDI-TOF mass spectrometry did not provide reliable identification using the Bruker Biotyper Database. Results and discussion: Genome-wide phylogeny and average nucleotide identity (ANI) values confirmed that the studied strains are clearly differentiated from their closest phylogenetic relative Mycobacterium aromaticivorans and other validly published Mycobacterium species (ANI ≤ 85.0%). The type strain CVI_P3T was further characterized by a polyphasic approach using both phenotypic and genotypic methods. Based on the phenotypic, chemotaxonomic and phylogenetic results, we conclude that strains CVI_P3T and CVI_P4 represent a novel species, for which the name Mycobacterium pinniadriaticum sp. nov. is proposed.

Resistome Analysis of Campylobacter jejuni Strains Isolated from Human Stool and Primary Sterile Samples in Croatia.

Campylobacteriosis represents a global health challenge due to continuously increasing trends of antimicrobial resistance in Campylobacter jejuni. C. jejuni can sometimes cause life-threatening and severe systematic infections (bacteremia, meningitis, and other extraintestinal infections) with very few antibiotics left as treatment options. Bearing in mind that C. jejuni is the predominant species in humans, in this paper, we present a study of the C. jejuni differences in antimicrobial resistance and genotype distribution between strains isolated from stool and primary sterile sites. We compared the genomic data obtained through whole genome sequencing (WGS) and phenotypic susceptibility data of C. jejuni strains. Once antimicrobial susceptibility testing of C. jejuni strains was carried out by the broth microdilution method for six of interest, results were compared to the identified genotypic determinants derived from WGS. The high rate of resistance to fluoroquinolones presented in this study is in accordance with national surveillance data. The proportion of strains with acquired resistance was 71% for ciprofloxacin and 20% for tetracycline. When invasive isolates were analysed separately, 40% exhibited MIC values of ciprofloxacin higher than the ECOFFs, suggesting a lower flouroquinolone resistance rate in invasive isolates. All isolates demonstrated wilde-type phenotype for chloramphenicol, erythromycin, gentamicin, and ertapenem. A special focus and review in this study was performed on a group of C.jejuni strains found in primary sterile samples. Apart from demonstrating a lower resistance rate, these isolates seem genetically more uniform, showing epidemiologically more homogenous patterns, which cluster to several clonal complexes, with CC49 being the most represented clonal complex.

Antimicrobial resistance profiles of human Brucella melitensis isolates in three different microdilution broths: the first multicentre study in Bosnia and Herzegovina.

OBJECTIVES: Brucellosis is a ubiquitous emergent bacterial zoonotic disease causing significant human morbidity in Bosnia and Herzegovina. So far, a high rate of resistant Brucella has been found worldwide. This study prospectively analysed the rates of resistance among human Brucella melitensis strains isolated in Bosnia and Herzegovina. METHODS: This study included 108 B. melitensis isolates from 209 patients diagnosed at five medical centres in Bosnia and Herzegovina. The resistance profiles of the B. melitensis isolates for the 13 most commonly used antimicrobials were studied in standard Brucella broth (BB) and cation-adjusted Mueller-Hinton broth (CAMHB) supplemented with 4% lysed horse blood or 5% defibrinated sheep blood. RESULTS: Of the 209 patients, B. melitensis blood cultures were positive for 111 (53.1%). Among the 108 isolates investigated, 91 (84.3%) were resistant to trimethoprim-sulfamethoxazole on BB, but not on either CAMHB. Nearly all isolates (>90%) were resistant to azithromycin on BB and both CAMHBs. CONCLUSION: We observed a high rate of B. melitensis resistance to azithromycin. The high rate of resistance to trimethoprim-sulfamethoxazole that we observed was related to BB, so an alternative broth should be used, such as the enriched CAMHBs in this study, for evaluating resistance to trimethoprim-sulfamethoxazole. Whole-genome sequencing studies are needed to understand the development of antimicrobial resistance in B. melitensis strains isolated from humans.

The presence of methicillin-resistant Staphylococcus aureus on large pig breeding farms in Croatia.

Methicillin-resistant Staphylococcus aureus (MRSA) have emerged worldwide and have become resistant to a variety of antibiotics. MRSA colonisation in pigs was first reported from the Netherlands in 2005, where pigs were implicated as a source of human MRSA infections (Voss et al., 2005). This paper presents the first report on the presence of MRSA on large pig breeding farms in Croatia, together with the determination of the mecA gene, the results of spa typing and susceptibility to commonly used antimicrobials. Dust samples (7-11 per farm) were collected from eight large pig farms in Croatia. Of the total 68 swabs, the mecA gene was detected in 24 isolates growing on the MRSA agar. All isolates were resistant to oxacillin, tetracycline and streptomycin, and susceptible only to vancomycin, while 92% of the strains were susceptible to ciprofloxacin. Genotyping of the MRSA strains was performed by spa typing, and revealed t011 (n = 17), t034 (n = 5) and t1451 (n = 2). The results presented here predict that MRSA is present on a large number of pig farms in Croatia.

Occurrence of Campylobacter jejuni in Gulls Feeding on Zagreb Rubbish Tip, Croatia; Their Diversity and Antimicrobial Susceptibility in Perspective with Human and Broiler Isolates.

Campylobacteriosis is the most common gastrointestinal bacterial disease in the European Union (EU). Wild birds are one of the natural reservoirs of these pathogens. In this study we tested cloacal swabs of 643 gulls captured on rubbish tip in Zagreb, Croatia for the presence of Campylobacter spp. and found 168 Campylobacter positive samples. We used multilocus sequence typing (MLST) to genotype 62 random C. jejuni isolates from gulls, 24 isolates from broiler caeca, 27 isolates from broiler neck skins and 23 human isolates. Altogether, we identified 44 different STs, from which 19 were newly described. Most of the new STs (14) originate from gulls. Although humans and broilers share the majority of STs and isolates from gulls are separated from these, there was one ST present in all three hosts: 45. Additionally antimicrobial susceptibility to six antimicrobials was performed on 123 C. jejuni strains isolated from broiler caeca (n = 22), neck skins of broilers (n = 20), gulls cloacal swabs (n = 50) and human faeces (n = 31) by the broth microdilution method. Results show lower resistance of gull isolates to NAL and CIP, while resistance to TET was as high as in human and broiler isolates.